Hexavalent chromium detoxification by haloalkaliphilic Nesterenkonia sp strain NRC-Y immobilized in different matrices

Abstract To develop a bioprocess for Cr(VI) detoxification in industrial effluents, a previously isolated potent Cr(VI) reducing haloalkaliphilic Nesterenkonia sp strain NRC-Y was immobilized and investigated for Cr(VI) detoxification. The immobilization matrices included; natural, modified natural, synthetic, and mixtures of natural and synthetic polymers. Among the tested matrices and immobilization approaches, strain NRC-Y cells encapsulated in 1.5% (w/v) amidated pectin beads exhibited the highest Cr(VI) reduction efficiency (47.02% of initial Cr(VI) concentration 150 mg/L after 20 h) followed by alginate (34.4%), alginate-PVA-chitosan (29.6%), alginate-PVA (25.1%), PVA-PVP (23.2%), and PVA (9.5%). Chromate reductase enzyme was poorly immobilized by covalent binding on the tested materials. Therefore, entrapment in amidated pectin was selected for further investigation and immobilization of both whole cells and partially purified chromate reductase. Operational stability study revealed that the immobilized cells were more efficient and stable than the immobilized chromate reductase, and the free cells. For instance, about 60%, 27.0% and 11.5% of these samples initial activities were retained after four successive batches, respectively. The temperature and pH optima for the immobilized cells were 35°C and 7.0, respectively. The pH and thermal stability of strain NRC-Y cells were significantly enhanced upon immobilization in amidated pectin beads by up to 2.3- and 1.4-fold, respectively. The developed immobilized biocatalyst was applied for Cr(VI) reduction in industrial effluent samples, and it completely reduced Cr(VI) within 4 and 8 h for effluents of initial Cr(VI) concentrations 10 and 30 mg/L, respectively. The developed immobilized biocatalyst is promising and has the potential for large-scale Cr(VI) detoxification application. Graphical Abstract


Introduction
Hexavalent chromium [Cr(VI)] is a notable pollutant resulting from its wide applications in a variety of industries, such as leather tanning, steel and automobile production, electroplating, fertilizers, paint pigment, mining, dye manufacturing in addition to nuclear wastes (Lyu et al. 2017;Chen and Tian 2021).In the environment, hexavalent [Cr(VI)] and trivalent [Cr(III)] are the most stable oxidation states of chromium (Chai et al. 2019).Cr(VI) is highly soluble and mobile; consequently, it readily spreads in polluted environments.It is also easily taken up by organisms by means of the sulfate transport system.Thus, Cr(VI) is well known carcinogenic and mutagenic agent in humans and animals.In addition, Cr(VI) causes plant roots damage, and this could inhibit plants growth (Chen et al. 2018;Chen and Tian 2021).In contrast, Cr(III) is immobile and is relatively nontoxic therefore Cr(VI) reduction to Cr(III) is an effective approach to detoxify Cr(VI) from polluted environments (Pradhan et al. 2017;Chen and Tian 2021).
Conventional methods for Cr(VI) remediation, such as chemical reduction, solvent extraction, and ion exchange techniques have significant drawbacks including high cost and production of toxic secondary pollutants as well as high energy consumption (Zeng et al. 2019;Chen and Tian 2021).Alternatively, bioremediation using microorganisms is as an effective and environment-friendly approach that is gaining increasing attention as a new remediation technology (Mala et al. 2015;Das et al. 2021).Several bacterial species have been reported for Cr(VI) reduction, such as Pseudomonas, Bacillus, Streptomyces, Pannonibacter and Cellulosimicrobium.These species produce chromate reductase enzymes which reduce the toxic Cr(VI) to non-toxic Cr(III) (Lin et al. 2012;Ibrahim et al. 2012;Karthik et al. 2017, Tan et al. 2020;Chen and Tian 2021).Enzymes have been recently used as potential biocatalysts in a large number of biotechnological applications including dairy products, pharmaceuticals, detergents, and environmental applications.Therefore, enzymes production and utilization are growing, and enzymes are becoming better alternatives to chemical catalysts with expected contribution of multibilliondollar in bio-industries.(Virgen-Ortiz et al. 2019;Monteiro 2020;Singla and Bhardwaj 2020;Silva et al. 2022).However, the bottle neck of enzymes and microorganisms application in Cr(VI) reduction may be the microbial survival.Unsheltered cells would be vulnerable to Cr(VI) toxicity particularly at high Cr(VI) concentrations.Furthermore, industrial application of free bacterial cells has several disadvantages such as the difficulties of biomass separation, and the toxicity of Cr(VI) that causes cells damage and enzymes inhibition (Wu et al. 2019;Monteiro et al. 2020).One of the main approaches applied to resolve such difficulties is to immobilize biocatalysts into solid supports.This can produce recoverable and stable heterogeneous biocatalysts, and is considered as a very powerful tool to improve biocatalysts stability and properties (Mohamed et al. 2014;Ishak et al. 2016;Esmaeili and Farrahi 2016;Wu et al. 2019).The use of immobilized microorganisms is superior to free cells in different applications as they exhibit enhanced stability and they could be simply recovered and reused (Kathiravan et al. 2010;Lu et al. 2018;Dey and Paul 2020;Silva et al. 2022).Furthermore, it preserves the natural environment of the intracellular enzyme, and therefore protecting it from inactivation during either immobilization or its subsequent application (Jobby et al. 2018;Neto et al. 2023).
A novel Cr(VI)-resistant haloalkaliphilic Nesterenkonia sp strain NRC-Y was previously isolated from hypersaline soda lakes and was characterized for its Cr(VI) bioreduction efficiency (Abo-Alkasem et al. 2023).Strain NRC-Y showed high Cr(VI) reduction activity (up to 800 mg/L within 24 h) under high salinity and alkalinity conditions.Furthermore, strain NRC-Y could grow and reduce Cr(VI) effectively in the presence of a variety of other heavy metals.Chromate reductase of Nesterenkonia sp strain NRC-Y was detected mainly in the bacterial cell membranes and cytoplasm (Abo-Alkasem et al. 2023).In this work, immobilization of Nesterenkonia sp strain NRC-Y whole cells and partially purified chromate reduction enzyme was investigated using various carriers including natural, modified natural and synthetic polymers as well as combination of natural and synthetic carriers.In addition, the most effective immobilized biocatalyst, in term of stability and activity, was applied for the detoxification of chromium in industrial effluent waste.The developed immobilized biocatalyst was able to completely reduce Cr(VI) in industrial effluent samples within 4-8 h.The developed immobilized biocatalyst is promising and has the potential for large-scale Cr(VI) detoxification application in contaminated water and industrial effluent.To the best of our knowledge, this is the first report about of Cr(VI) detoxification by immobilized Nesterenkonia sp strain NRC-Y in amidated pectin beads.

Microorganism and culture conditions
Haloalkaliphilic Nesterenkonia sp strain NRC-Y used in this study was recently isolated from hypersaline soda lakes, located in Wadi Natrun Valley in northern Egypt.The strain was propagated under the previously developed optimum culture conditions (Abo-Alkasem et al. 2023).All materials used in this study were of high purity grade.

Large scale preparation of partially purified chromate reductase
Loopful from agar plate culture of Nesterenkonia sp strain NRC-Y was transferred to five 250 mL Erlenmeyer flasks containing 50 ml of the alkaline medium (pH 10.5).This medium contained (g/L): 5 g casamino acids, 5 g peptone, 5 g yeast extract, 10 g glucose, 100 g NaCl, 10 g Na 2 CO 3 , 300 lL trace elements solution supplemented with 2 mM Cr(VI) (Abo-Alkasem et al. 2023).The cultures were incubated overnight at 40 C in shaking incubator (150 rpm) (to mid logarithmic phase).They were then used to inoculate (5%) five liters medium distributed in five 2-liter flasks.Afterwards, the cultures were incubated at 40 C and 150 rpm for 24 h.After the incubation period, the cells were collected by centrifugation at 10,000 rpm for 15 min in a cooling centrifuge.The obtained cell biomass was washed thrice with Tris buffer (50 mM; pH 7.0) and was re-suspended in 200 ml Tris buffer.Part of the biomass was broken by sonication using an Ultrasonic Probe (Omni 400, UK) with amplitude of 35% at 50 W with 5 s pulses and 5 s offmode for 15 min.The obtained cells lysate (CL) was then centrifuged at 15,000 rpm for 20 min at 4 C and the membranous fraction was collected, resuspended in Tris buffer (50 mM; pH 7.0) and used as partially purified chromate reductase enzyme.

Immobilization of chromate reductase and bacterial whole ells in variety of matrices
Chromate reductase immobilization by covalent binding Partially purified chromate reductase enzyme of Nesterenkonia sp NRC-Y was covalently immobilized on three different matrices as the following: (i) treated carrageenan-calcium pectinate (Car-CPT) beads.Treated carrageenan-calcium pectinate (Car- CPT) beads were prepared as previously reported by Wahba (2021).Afterwards, 1.0 gm beads was suspended in 5 ml partially purified chromate reductase solution (equivalent to 1.0 gm cells) prepared in Tris-HCL buffer (50 mM, pH 7).This mixture was kept overnight at 4 C to allow covalent binding between the activated Car-CPT carrier through the free aldehyde groups and the free amino acids of the enzyme.Thereafter, the beads were washed several time with the Tris buffer to rinse any unbound proteins, followed by assay of Cr(VI) reduction activity of the immobilized enzyme as shown below.
(ii) Soy protein isolate (SPI) activated carrageenan (Car) beads.Soy protein isolate (SPI) activated carra- geenan (Car) beads were prepared as previously reported by Wahba (2022).Afterwards, 1.0 gm beads was suspended in 5 ml partially purified chromate reductase solution (equivalent to 1.0 gm cells).This mixture was kept overnight at 4 C to allow covalent binding between the activated SPI-Car carrier via the free aldehyde groups and the free amino acids of the enzyme.Thereafter the beads were washed several times with 50 mM Tris buffer (pH 7) to rinse any unbound proteins, followed by assay of the Cr(VI) reduction activity of the immobilized enzyme.
(iii) Egg-white protein (EWP) activated gellan gum (GG) beads.Egg-white protein (EWP) activated gellan gum (GG) beads were prepared according to Wahba (2020).Afterwards, 1.0 gm beads was suspended in 5 ml partially purified chromate reductase solution (equivalent to 1.0 gm cells) prepared in Tris-HCL buffer (50 mM, pH 7).This mixture was kept overnight at 4 C to allow covalent binding between the activated EWP-GG carrier through the free aldehyde groups and the free amino acids of the enzyme.Thereafter, the beads were washed several time with the Tris buffer to rinse any unbound proteins, followed by assay of Cr(VI) reduction activity of the immobilized enzyme as shown below.

Immobilization of chromate reductase and whole cells in alginate beads
Partially purified chromate reductase and whole cells of Nesterenkonia sp NRC-Y were immobilized by entrapment in Ca-alginate beads according to Woodward (1988).Sodium alginate was solvated in distilled water to give final concentrations of 1, 2 and 3%.Afterwards, 2 ml of the enzyme preparation and whole cells suspension (equivalent to 1.0 gm cells) were mixed with 10 ml of each alginate solutions.The alginate mixtures were made into beads by dropping the alginate-cell/enzyme mixtures in 0.2 M CaCl 2 as cross-linking agent.The beads were retained for further 2 h before collection.They were then washed twice with 50 mM Tris buffer (pH 7), collected, and kept in the Tris buffer.Afterwards, the Cr(VI) reduction activities of the immobilized cells and immobilized enzyme were determined.

Immobilization of chromate reductase and whole cells in alginate-PVA-chitosan
The whole cells and partially purified chromate reductase of Nesterenkonia sp NRC-Y were immobilized by entrapment into gel matrices consisting of three polymers.(polyvinyl alcohol (PVA), alginate, and chitosan.)The matrices were prepared in two stages as the following procedures (Zhou et al. 2010;Pal et al. 2013;Dey and Paul 2015): (1) PVA (1.0 g) was suspended in 20 ml of sterile distilled water and was heated in boiling water bath till complete dissolution.Afterwards, 0.16 g of sodium alginate was introduced, and the mixture was further heated in boiling water bath till homogeneous solution was attained.The PVA-alginate solutions were cooled prior to the addition of 5 ml of the Nesterenkonia sp NRC-Y whole cell suspension or chromate reductase preparation (equivalent to 1.0 gm cells); (2) Alginate-PVA-chitosan gel composite: Chitosan (0.25%, w/v) was first suspended in acetic acid solution (5%) and dispersed ultrasonically for 30 min using Ultrasonic Probe (Cole-Parmer, USA) with amplitude of 45% at 50 W with 5 s pulses and 5 s offmode.Afterwards, CaCl 2 solution was introduced to the prepared chitosan solution to a final concentration of 2.0% (w/v).Finally, the immobilized cell/enzyme beads were prepared by extruding the alginate-PVAwhole cells or the alginate-PVA-enzyme drop-wisely via a 10 mL syringe with a 19-gauge needle into 200 mL chilled chitosan-CaCl 2 solutions and kept overnight for hardening at 4 C. Deactivated cells and chromate reductase enzyme (boiled for 15 min) were immobilized using the same procedures as control experiment.
(3) Treatment with glutaraldehyde: The beads were rinsed with sterile saline solution and were divided into parts.A part was further treated with 0.5% glutaraldehyde solution for 2 h to allow crosslinking of the chromate reductase and whole cells within the alginate-PVA-chitosan beads.Then the beads of immobilized whole cells and immobilized enzyme were washed several times with sterile distilled water and twice with 50 mM Tris buffer (pH 7).The Cr(VI) reduction activity of the developed immobilized whole cells and immobilized chromate reductase was determined.
Immobilization of chromate reductase and whole cells in alginate-PVA PVA (0.2 g) was suspended in 20 ml of sterile distilled water and was heated in boiling water-bath till complete dissolution of the PVA (1%).Afterwards, 0.4 g of sodium alginate (2%) was introduced and the mixture was further heated in boiling water bath till obtaining homogeneous solution (Pal et al. 2013).The PVA-alginate solutions were cooled prior to the addition of 5 ml of the Nesterenkonia sp NRC-Y whole cell suspension and partially purified chromate reductase preparation (equivalent to 1.0 gm cells).The cells-PVA-alginate and the enzyme-PVA-alginate mixtures were then dropped via a 10 mL syringe with a 19-gauge needle into 200 mL chilled 2% CaCl 2 solutions and were kept overnight for hardening at 4 C. Deactivated cells and chromate reductase (boiled for 15 min) were immobilized using the same procedures as controls.Afterwards, the formed spherical beads of immobilized whole cells and immobilized enzyme were washed several times with sterile distilled water and twice with 50 mM Tris buffer (pH 7).The Cr(VI) reduction activity of the developed immobilized whole cells and immobilized chromate reductase was determined.

Immobilization of chromate reductase and whole cells in amidated pectin
The partially purified chromate reductase and whole cells suspensions of Nesterenkonia sp NRC-Y (equivalent to 1.0 gm cells) were mixed with amidated pectin solutions so that the final amidated pectin concentration was 1.5% (w/w).The mixtures were stirred for at least 2 h at room temperature.Afterwards, the mixtures were extruded via a 10 mL syringe with a 19gauge needle into 5% CaCl 2 solution in order to acquire the gel beads.The formed spherical beads of immobilized whole cells/enzyme were then washed several times with sterile distilled water and twice with 50 mMTris buffer (pH 7).Part of the formed spherical beads was further treated with 0.5% glutaraldehyde solution for 2 h to allow crosslinking of the chromate reductase and whole cells within the amidated pectin beads.Thereafter, the Cr(VI) reduction activity of the developed immobilized whole cells and immobilized chromate reductase was determined.
Immobilization of chromate reductase and whole cells in PVA and PVA-PVP Immobilization of chromate reductase and whole cells in PVA and PVA-PVP was carried out according to Dey and Paul (2015) with some modifications.Either the partially purified chromate reductase or cell suspension was mixed with a cooled melted polyvinyl alcohol (PVA) solution so that the final PVA concentration was 10% (w/v).The mixtures were left at room temperature for at least 2 h.Afterwards, they were extruded via a syringe thin needle into 1 M boric acid/borax buffer (pH 7.6) in order to acquire the gel beads.For encapsulation in polyvinylalcohol-polyvinylpyrrolidone (PVP), either the chromate reductase enzyme or cell suspension (equivalent to 1.0 gm cells) was mixed with a PVA-PVP solution so that the final PVA-PVP concentrations were 10% and 1%, respectively.The mixtures were left at room temperature for at least 2 h.Afterwards, they were extruded via a 10 mL syringe with a 19-gauge needle into 1 M boric acid/borax buffer (pH 7.6) in order to acquire the gel beads.The formed spherical beads of immobilized whole cells/enzyme were then washed several times with sterile distilled water and twice with 50 mM Tris buffer (pH 7).The Cr(VI) reduction activity of the developed immobilized whole cells and immobilized chromate reductase was determined.

Assay of chromate reduction by the immobilized cells/enzyme
For assaying the Cr(VI) reduction activity of the immobilized cells or enzyme, 1.0 gm of the beads was suspended in 10 ml the reaction substrate [50 mM Tris buffer (pH 7) containing 150 mg/L Cr(VI), 50 mg/L Cu 2þ , 1.0% glycerol], The reaction mixtures were incubated in shaking incubator at 35 C for 20 h.Heat killed immobilized cells or enzyme, free cells and free enzyme were used as controls (Wu et al. 2019;Abo-Alkasem et al. 2023).Thereafter, samples were centrifuged and the residual Cr(VI) concentration was determined in the reactions supernatant following 1,5-diphenylcarbazide (DPC) method (Bartlett and James 1996;Abo-Alkasem et al. 2023).

Determination of Cr (VI) concentration
The concentration of Cr (VI) was measured calorimetrically using 1,5-diphenylcarbazide (DPC) method (Bartlett and James 1996;Abo-Abo-alkasem et al. 2023).Briefly, 300 lL samples were added to 5 ml of distilled water in glass test tubes.Afterwards, 1.0 mL of DPC solution (0.25% [w/v], prepared in acetone) was added followed by the addition of 50 lL phosphoric acid (85%).After incubating the reaction mixtures for 10 min at room temperature, the developed colour absorbance was measured at 540 nm.The calibration curve was developed using K 2 Cr 2 O 7 at concentrations between 10 to 300 lgmL À1 .

Properties of immobilized whole cells and chromate reductase
Operational stability of the free and immobilized cells/enzyme The operational stability and reusability of free and immobilized cells/enzyme in amidated pectin beads was investigated via assaying the respective preparations in successive batches in 10 ml of the standard assay mixture containing 150 mg/L Cr(VI).The samples were incubated in a shaking incubator (rpm 120) at 35 C for 20 h.After processing each batch, the free and immobilized cells/enzyme were removed from the reaction medium and were washed twice with 50 mM Tris buffer (pH 7) to remove any remaining substrate or products.They were then introduced into fresh reaction mixtures for a new cycle.At the end of each cycle, the residual Cr(VI) concentration was determined using DPC and the Cr(VI) reduction was calculated and expressed relative to the initial activity.

Effect of temperature on activity of immobilized whole cells
The Cr(VI) reduction activities of free and immobilized cells in amidated pectin beads were measured at various temperatures ranging from 25 C to 50 C under the standard assay conditions.In addition, to investigated the influence of temperature on cell stability, both free and immobilized cells were incubated at various temperatures ranged from 25 C to 50 C for 1 h in a shaking water bath, prior to measurement of the residual Cr(VI) reduction activity using the standard assay conditions.The relative activity of the free and immobilized cells was calculated in comparison to untreated samples.

Effect of pH on the activity of immobilized whole cells in amidated pectin
The influence of pH on Cr(VI) reduction activity of free and immobilized cells in amidated pectin beads was studied by measuring the Cr(VI) reduction activity at varying pH values which ranged from pH 4 to 10 at 35 C using appropriate buffers.The used buffers were sodium acetate (pH 5.0 and 6.0), sodium phosphate (pH 7.0 and 8.0), and glycine-NaOH buffer (pH 9.0 and 10.0) at molarity of 50 mM.Furthermore, to investigate the influence of pH on cell stability, free and immobilized whole cells were incubated in buffers at the designated pH for 24 h at room temperature, and then the residual activity was measured under the standard assay conditions.

Application of the developed biocatalyst for Cr(VI) detoxification in industrial effluent
Chromium containing industrial effluent samples were collected from different leather tanning factories in El-Robieky City for Leather Industries, located in Badr city (Egypt).The Cr(VI) content of the collected industrial effluents was determined.Cr(VI) bioremoval from the leather industrial effluent samples was carried out using the developed biocatalyst, Nesterenkonia sp NRC-Y cells immobilized in amidated pectin beads.Briefly, 3.0 gm of immobilized cells beads were suspended in 20 mL of the industrial effluent and incubated at 35 C in shaking incubator (120 rpm).Then, samples were withdrawn from the reaction 1h interval for measurement of the residual Cr(VI).

Statistical analysis
The experiments and assays were carried out in triplicate.The standard deviations for each experiment were estimated using Microsoft Excel.

Results and discussion
Large scale preparation of whole cells and partially purified chromate reductase Ten liters of Nesterenkonia sp NRC-Y culture were prepared.The cell biomass was then collected, washed and a part of it was broken by sonication using an Ultrasonic Probe (Figure S1).Afterwards, the membranous fraction was prepared and resuspended in 50 mM Tris-HCl buffer (pH 7) as described above.Both Nesterenkonia sp NRC-Y whole cells and the partially purified chromate reductase were immobilized in a variety of matrices.

Immobilization of chromate reductase by covalent binding
Partially purified chromate reductase of Nesterenkonia sp strain NRC-Y was covalently immobilized on three different matrices including carrageenan-calcium pectinate (Car-CPT), soy protein isolate (SPI) activated Car, and egg-white protein (EWP) activated gellan gum (GG) beads (Figure S2).The results presented in Table 1 indicted that the chromate reductase enzyme was poorly immobilized on the three matrices with the highest reduction activity achieved by Car-CPT (12.5%), followed by SPI-Car (11.0%) and EWP-GG (8.0%).The low activity of immobilized chromate reductase activity may be due to the immobilization induced inactivation and/or weak binding between the enzyme and the carriers (Ibrahim et al. 2012;Wahba 2022).

Immobilization of chromate reductase and whole cells in alginate beads
The partially purified chromate reductase and whole cells of Nesterenkonia sp NRC-Y were immobilized by entrapment approach in alginate beads.Alginate is a natural polymer and its concentration was varied among 1-3%.The results presented in Table 2 indicated that both the whole cells and partially purified chromate reductase were entrapped successfully in alginate beads.Maximal Cr(V) reduction was shown using 2.0% alginate with Cr(VI) reduction of 43.8% and 34.4%, for immobilized whole cells and enzyme, respectively.

Immobilization of chromate reductase and whole cells in alginate-PVA-chitosan
The whole cells and partially purified chromate reductase of Nesterenkonia sp NRC-Y were immobilized by entrapment into alginate-PVA-chitosan gel composite, as a mixture of synthetic and natural polymers.Addition of synthetic polymers such as PVA to natural polymers for cell/enzyme immobilization could enhance the mechanical strength of the carriers (Wu et al. 2019;Dey and Paul 2020).Furthermore, part of the formed immobilized cells/enzyme beads was treated with glutaraldehyde solution to allow crosslinking of the chromate reductase and whole cells within the alginate-PVA-chitosan beads (Zhou et al. 2010).The Cr(VI) reduction activity of the developed immobilized whole cells/enzyme was determined The immobilization matrices were treated carrageenan-calcium pectinate (Car-CPT), soy protein isolate (SPI) activated carrageenan (Car), and eggwhite protein (EWP) activated gellan gum (GG) beads.Results represented means of three separate experiments, and the standard deviations were in the range of 2.2-3.0%.
(Figure S3).The results shown in Table 3 indicated that both whole cells and partially purified chromate reductase were successfully entrapped within the alginate-PVA-chitosan composite beads as 29.6% and 25.1% Cr(VI) reduction percents were achieved, respectively.However, treating the beads with glutaraldehyde as a crosslinking agent caused severe decrease in Cr(VI) reduction activity of both immobilized cells and chromate reductase enzyme to 3.0% and 6.9%, respectively.Inhibition of immobilized enzymes by glutaraldehyde is mostly due to the crosslinking in the amino acids of the enzymes active sites (Zhou et al. 2010).

Immobilization of chromate reductase and whole cells in alginate-PVA composite
The whole cells and chromate reductase enzyme of Nesterenkonia sp NRC-Y were immobilized by entrapment into alginate-PVA gel composite, as a mixture of natural (alginate) and synthetic (PVA) polymer (Figure S4).The results shown in Table 4 indicated that both whole cells and chromate reductase were successfully entrapped within the alginate-PVA composite.However, the immobilized whole cells showed higher Cr(VI) reduction activity than immobilized chromate reductase (42.7% and 25.1%, respectively).Addition of the synthetic polymers such as PVA to the natural polymers for cell/enzyme immobilization can improve the mechanical strength of the carriers (Wu et al. 2019;Dey and Paul 2020).

Immobilization of whole cell and chromate reductase enzyme in amidated pectin
The whole cells and partially purified chromate reductase of Nesterenkonia sp NRC-Y were immobilized by entrapment in 1.5% amidated pectin as a modified natural polymer, and the Cr(VI) reduction efficiency was evaluated (Figure S5).Furthermore, part of the immobilized cells/enzyme beads was treated with glutaraldehyde solution to allow crosslinking of the chrome reductase and whole cells within the amidated pectin beads.The chromate detoxification efficiency of the developed immobilized whole cells/enzyme was determined.The results presented in Table 5 indicated that both whole cells and chromate reductase were successfully entrapped within the amidated pectin beads as 47.02% and 58.4% Cr(VI) reduction percents were achieved, respectively.However, treating the beads with glutaraldehyde as a crosslinking agent caused drastic decrease in the Cr(VI) reduction activity of both immobilized cells and immobilized chromate reductase to 7.2% and 11.9%, respectively.Inhibition of the immobilized enzyme by glutaraldehyde is mostly due to the crosslinking in the amino acids of the enzymes active sites (Zhou et al. 2010).
Immobilization of chromate reductase and whole cells in PVA and PVA-PVP Furthermore, the whole cells and the partially chromate reductase of Nesterenkonia sp NRC-Y were immobilized by entrapment in synthetic polymers including PVA and PVA-polyvinylpyrrolidone (PVP) composite.The results presented in Table 6 indicated that both immobilized cells and immobilized enzyme showed very low Cr(VI) reduction activity of 18.9 and 23.2% using PVA and 16.3% and 9.5% using PVA/PVP composite, respectively.The low Cr(VI) reduction by PVA beads might be due to constrains related to chromate and electron donor diffusion into the beads (Dey and Paul 2015).

Comparison of the tested carriers for whole cells/chromate reductase immobilization
The use of bacterial free whole cells and cell-free extracts (CFE) for chromate reduction has certain disadvantages, such as low mechanical strength, difficulties in separation of biomass from effluent, and cells damage owing to toxicity.One of the main approaches applied to resolve these difficulties is to immobilize the biocatalysts onto solid supports so as to produce recoverable and stable heterogeneous biocatalysts.Moreover, immobilization is considered as a very powerful tool to improve biocatalysts stability and properties (Dey and Paul 2020;Wang et al. 2020).
In various applications, it is advantageous to use immobilized microorganisms, rather than free cells, as this enhances the stability of the biocatalysts and facilitates their recovery and reuse.Furthermore, it preserves the natural microenvironment of intracellular enzymes.Accordingly these enzymes would be protected from inactivation during the immobilization process and also during their subsequent use in continuous bioremediation processes.Increased plasmid stability compared to free cells is also an added advantage in this process (Jobby et al. 2018, Dey andPaul 2020;Wang et al. 2020).However, the selection of immobilization matrices and approaches are key parameters in the successful application of immobilized microorganisms and enzymes.Therefore, nine different materials were investigated as carriers for immobilizing the whole cells and the partially purified chromate reductase of Nesterenkonia sp NRC-Y.These included; a natural polymer (alginate); a modified natural polymer (amidated pectin); synthetic polymers (PVA, PVA-PVP), and mixtures of natural and synthetic polymers (Alginate-PVA and alginate-PVA-Chitosan).In addition, other three carriers were investigated for covalent immobilization of partially purified chromate reductase enzyme including carrageenan-calcium pectinate (Car-CPT), soy protein isolate (SPI) activated Car, and egg-white protein (EWP) activated gellan gum (GG) beads.Table 7 summarized the results of the immobilization experiments using whole cells and partially purified chromate reductase of Nesterenkonia sp NRC-Y in the tested carriers.The results indicated that amidated pectin hydrogel (modified natural material) was the most suitable support for immobilization of both whole cell and enzyme by entrapment approach as it achieved Cr(VI) reduction precents of 47.02% and 58.4%, respectively, followed by alginate (34.4%), alginate-PVA-chitosan (29.6%), alginate-PVA (25.1%),PVA-PVP (23.2%), and PVA (9.5%).The low Cr(VI) reduction by synthetic polymers including PVA and PVA-PVP beads might be due to constrains related to chromate and electron donor diffusion into the beads (Dey and Paul 2014).In addition, the chromate reductase enzyme was poorly immobilized by covalent  Results represented means of three separate experiments, and standard deviations were in the range of 2.9-3.7%.
binding on Car-CPT, SPI-Car and EWP-GG.This was evidenced by the recorded Cr(VI) reduction percents which amounted to only 2.5%, 11.0 and 8.0%, respectively.The low activity of immobilized chromate activity may be due to the immobilization induced inactivation and/or weak binding between the enzyme and the carriers through the cross-linker (Ibrahim et al. 2012;Wahba 2022).
The Cr(VI) removal capacity of amidated pectin immobilized cells bio-composite (3.53 mg À1 h À1 ), was more efficient than other immobilized Cr(VI) reducing bacterial cells (Table S1).On another occasion, Pal et al. (2013) reported that PVA-alginate was a more effective matrix for the immobilization of Bacillus sphaericus AND 303 than Ca-alginate, PVA-borate, agarose and agar-agar.The PVA-alginate immobilized cells reduced 87.5% of 20 mM Cr(VI) in 24 h at a removal rate of 0.245 mg À1 h À1 .However, Arthrobacter sp.SUK 1205, which was immobilized in Ca-alginate, was better than that its PVA-alginate, PVA-borate and Ba-alginate immobilized analogues as it showed complete reduction of 100 mM Cr(VI) within 24 h with Cr(VI) removal rate of 1.14 245 mg À1 h À1 (Dey and Paul 2015).Recently, Bacillus sp strain CRB-7 cells, which were immobilized with 3% sodium alginate and 5% humic acid, showed the highest Cr(VI) reduction efficiency of 2.080 mg À1 h À1 (Wu et al. 2019).Further comparisons with other immobilized Cr(VI) reducing bacterial cells are shown in Table S1.
The efficiency of the amidated pectin immobilized Nesterenkonia sp strain NRC-Y could be due to several factors, such as the enhanced metabolic activity and the better protection provided to the bacterial cells which eventually led to better Cr(VI) reduction efficiency.Furthermore, higher reaction rates of the immobilized Nesterenkonia sp strain NRC-Y could have been caused by the high local cells density and cells stabilization.On the other hand, improper immobilization matrices could have resulted in low diffusion of the electron donor or substrate and could have also induced less cells stabilization.Thus, the Cr(VI) reduction rates were decreased (Wu et al. 2019).Therefore, amidated pectin hydrogel was selected for immobilization and further investigation of both whole cells and partially purified chromate reductase of Nesterenkonia sp NRC-Y.Pectins are a family of natural biopolymers that are purified from the plant cell wall.Pectins are composed of an anionic polysaccharide backbone of a-1,4linked ᴅ-galacturonic acids.These acids are partially substituted by methyl esters and/or carboxamide groups.Pectins differ according to their degree of esterification (DE) and/or amidation (DA) (Huynh et al. 2017).Amidated pectins were applied as scaffolding drug delivery systems, owing to their versatility targeting specific sites and releasing rates (Sriamornsak 2011).However, application of pectins as carriers for immobilization of microorganisms is still relatively unexplored (Bepeyeva et al. 2017;Barrera et al. 2020).Therefore, to the best of our knowledge, this is the first report for application of amidated pectin as carrier for immobilization of whole cells/enzyme for Cr(VI) bioreduction and bioremediation application.

Properties of immobilized whole cells/chromate reductase in amidated
Operational stability of immobilized whole cells/chromate reductase In order to compare between the Cr(VI) reduction efficiencies of the amidated pectin immobilized whole cells and the amidated pectin immobilized partially purified chromate reductase of Nesterenkonia sp NRC-Y, the operation stabilities of both immobilized systems were investigated by the determination of the Cr(VI) reduction efficiency in six successive cycles.The results presented in Figure 1 revealed that the immobilized whole cells were more efficient and stable than the immobilized chromate reductase as they retained about 60% and 46% of their initial activity after four and five successive cycles, respectively.On the other hand, the immobilized chromate reductase enzyme retained only 27.0% and 15.4% respectively.In addition, the free whole cells only 11.5% and 2.6% of their initial activity after four and five successive cycles, respectively.This revealed the more toxic effect of Cr(VI) on the free Nesterenkonia sp NRC-Y cells.The higher stability and efficiency of the immobilized whole cells, as compared to the immobilized chromate reductase, is mostly due to the fact that the whole cell preserves the natural intracellular fairly stable microenvironment, and this would better stabilize the intracellular chromate reductase enzyme of Nesterenkonia sp NRC-Y (Wang et al. 2020).Furthermore, cells encapsulation within amidated pectin provided protection from the Cr(VI) toxicity (Tripathi and Garg 2013).These results were relatively similar to those of the Ba-alginate immobilized Arthrobacter sp.SUK 1201 cells which showed 60% Cr(VI) reduction (initial Cr(VI) concentration 100 mM) in the fourth cycle (Dey and Paul 2014).
Bacillus sp CRB-7 immobilized in alginate-humic acid could also be reused for Cr(VI) reduction up to four cycles with high efficiency (Wu et al. 2019).Noteworthy, Nesterenkonia sp NRC-Y whole cells immobilized in amidated pectin were more efficient and stable than Bacillus cereus cells immobilized in alginate, agarose and polyacrylamide which showed Cr(VI) removal of 23.5%, 12.5% and 15% in the third cycle, respectively (Tripathi and Garg 2013).The ability of the amidated pectin immobilized whole cells to be recycled at least four times clearly indicated that Nesterenkonia sp NRC-Y is a promising biosystem for the development of a continuous process for treatment of Cr(VI) contaminated effluents.Therefore, it was selected for further investigation and application.
Effect of temperature on the activity of immobilized whole cells For determination of the optimum temperature for Cr(VI) reduction by the Nesterenkonia sp NRC-Y whole cells immobilized in amidated pectin beads, the activity was measured at different temperatures ranging from 25 to 50 C.The results illustrated in Figure 2(A), indicated that the optimum Cr(VI) reduction activity was seen at 35 C for both free and immobilized cells.However, by increasing the reaction temperature the relative activities of the immobilized cells were much higher than free cells.At temperatures 40 C and 45 C, the relative activities of the cells entrapped in amidated pectin beads were higher than the activities observed for free cells by approximately 27% and 29%, respectively.In addition, the thermal stability of free and immobilized whole cells was studied (Figure 2(B)).The enzyme activities of free and immobilized whole cells were 92-100% of their initial activities after incubation at 25-30 C for 1h.However, the immobilized cells retained more activity than the free cells after being treated at higher temperatures (35 to 50 C) Entrapping the Nesterenkonia sp NRC-Y whole cells in amidated pectin beads enhanced their thermostability, as compared to the free cells, by approximately 1.1-to 2.3-fold at temperatures ranging from 35 C to 50 C.The higher thermal stability of chromate reductase in case of the immobilized cells is primarily regarded to the fact that the enzyme is maintained within its natural intracellular microenvironment.Moreover, the immobilized cells are further protected within the amidated pectin beads (Ibrahim et al. 2012).Effect of pH on the activity of immobilized whole cells effect of pH on Cr(VI) reduction by the free and amidated pectin immobilized Nesterenkonia sp NRC-Y cells was determined by measuring the Cr(VI) reduction activity at varying pH values ranging from pH 4 to 10 at 35 C. Maximum Cr(VI) reduction activity was observed at pH 7 for both free and immobilized cells with a characteristic pH curve (Figure 3(A)).However, the relative activities of the immobilized cells were slightly higher than the free cells.At pH 9 and 10, the relative activities of the immobilized cells were 58.9% and 32.0%, respectively, whereas the relative activities of the free cells were 47.0% and 15.2% respectively (Figure 3(A)).The enzymatic activities of free and immobilized Nesterenkonia sp NRC-Y cells were stable for at least 24 h in a broad pH range from 5 to 10 (Figure 3(B)).In addition, immobilization of Nesterenkonia sp NRC-Y cells in amidated pectin beads enhanced their pH stability by about 1.1 to 1.4-fold.The high pH stability of chromate reductase for both free and immobilized cells is primarily regarded to the fact that the enzyme is maintained within its natural intracellular microenvironment.Moreover, the immobilized cells are further protected within the amidated pectin beads (Ibrahim et al. 2012).However, the decline of Cr(VI) reduction at extreme low/high pH is mostly regarded to of cell viability.

Application of the developed bioprocess in Cr(VI) detoxification in industrial effluent
In order to extend this work to environmental applications, the developed biocatalyst was used to treat industrial effluent containing Cr(VI) as a major component.Chromium containing industrial effluent waste samples were collected from different leather tanning factories in El-Robieky City for Leather Industries (Badr City, Egypt) (Figure S6).Chemical analysis of the different effluent samples indicated that the samples were rich in Cr(VI) with average concentration of 10-30 mg/L depending on the samples source.Detoxification of Cr(VI) in the collected leather industrial effluent samples was carried out using the developed biocatalyst, Nesterenkonia sp NRC-Y cells immobilized in amidated pectin hydrogel beads.The results illustrated in Figure 4, indicated that Cr(VI) reduction was completed in 4 and 8 h for effluent samples with initial Cr(VI) concentrations of 10 and 30 mg/L, respectively.This result clearly indicated that the developed immobilized biocatalyst is promising and has the potential for large-scale Cr(VI) detoxification application contaminated water and industrial effluents.

Conclusion
Twelve different materials have been investigated as carriers for the immobilization of partially purified chromate reductase and the whole cells of Nesterenkonia sp NRC-Y.These included; natural polymers, modified natural polymers, synthetic polymers, and mixture of natural and synthetic polymers.Among the tested carriers and immobilization approaches, Nesterenkonia sp NRC-Y cells encapsulated in amidated pectin beads showed highest Cr(VI) reduction efficiency.Operational stability study revealed that immobilized whole cells were more efficient and stable than both the immobilized chromate reductase and the free cells.The ability of the amidated pectin immobilized Nesterenkonia sp NRC-Y cells to be recycled at least four times clearly proved that the developed biosystem is promising for the development of a continuous process for treatment of Cr(VI) containing industrial effluents.Furthermore, the pH and thermal stability of Nesterenkonia sp NRC-Y cells were significantly enhanced upon immobilization in amidated pectin beads.This is the first report about the application of amidated pectin as carrier for immobilization of whole cells/enzyme for Cr(VI) reduction and bioremediation application.It is also the first

Figure 1 .
Figure 1.Operation stability of the free Nesterenkonia sp NRC-Y whole cells, their immobilized analogues, and the immobilized partially purified chromate reductase.Immobilization was accomplished by entrapment in amidated pectin.Results represent the means of three separate experiments, and the standard deviations are shown as error bars.

Figure 2 .
Figure 2. Temperature profiles (A) and thermal stability (B) of the free Nesterenkonia sp NRC-Y whole cells and their amidated pectin immobilized analogues.Results represent the means of three separate experiments, and the standard deviations are shown as error bars.

Figure 3 .
Figure 3.Effect of pH on Cr(VI) reduction activity (A) and pH stability (B) of the free Nesterenkonia sp NRC-Y whole cells and their amidated pectin immobilized analogues.Results represent the means of three separate experiments, and the standard deviations are shown as error bars.
report of Cr(VI) detoxification by immobilized Nesterenkonia sp NRC-Y.The developed immobilized biocatalyst was able to completely reduce Cr(VI) in industrial effluent samples within 4-8 h.The developed immobilized biocatalyst is promising and has the potential for large-scale Cr(VI) detoxification applications in contaminated water and industrial effluents.Future work includes investigation of different approaches to enhance the mechanical strength and properties of the amidated pectin immobilized cells, investigation of large-scale application of the developed biocatalyst, and development of a continuous process.

Figure 4 .
Figure 4. Hexavalent chromium Cr(VI) detoxification in the industrial effluent samples Fulfilled by the Nesterenkonia sp NRC-Y whole cells immobilized in amidated pectin beads.The experiments were carried at 35 C in shaking incubator (120 rpm) using effluents with two different initial Cr(VI) concentrations, 10 mg/L and 30 mg/L.Results represent the means of three separate experiments, and the standard deviation are shown as error bars.

Table 2 .
Immobilization of Nesterenkonia sp NRC-Y whole cells and partially purified chromate reductase in alginate beads.
Results represented means of three separate experiments, and standard deviations were in the range of 2.5-3.6%.Table 1.Cr(VI) reduction by the covalently immobilized Nesterenkonia sp NRC-Y partially purified chromate reductase.

Table 3 .
Immobilization of Nesterenkonia sp NRC-Y whole cell and partially purified chromate reductase in alginate-PVA-chitosan composite.

Table 4 .
Immobilization of Nesterenkonia sp NRC-Y whole cell and partially purified chromate reductase enzyme in amidated pectin beads.

Table 5 .
Immobilization of Nesterenkonia sp NRC-Y whole cell and partially chromate reductase in alginate and alginate-PVA composite.

Table 6 .
Immobilization of Nesterenkonia sp NRC-Y whole cells and partially purified chromate reductase in PVA and PVA/PVP.

Table 7 .
Comparison of all of the tested carriers for immobilization of Nesterenkonia sp NRC-Y whole cells and partially purified chromate reductase.