Immobilization of Pseudomonas aeruginosa static biomass on eggshell powder for on-line preconcentration and determination of Cr (VI)

Abstract In the present work, a novel continuous flow system (CFS) is developed for the preconcentration and determination of Cr (VI) using Pseudomonas aeruginosa static biomass immobilized onto an effective and low-cost solid support of powdered eggshells. A mini glass column packed with the immobilized biosorbent is incorporated in a CFS for the preconcentration and determination of Cr (VI) from aqueous solutions. The method is based on preconcentration, washing and elution steps followed by colorimetric detection with 1,5-diphenyl carbazide in sulphuric acid. The effects of several variables such as pH, retention time, flow rate, eluent concentration and loaded volume are studied. Under optimal conditions, the CFS method has a linear range between 10 and 100 μg L-1 and a detection limit of 6.25 μg L-1 for the determination of Cr (VI). The sampling frequency is 10 samples per hour with a preconcentration time of 5 mins. Furthermore, after washing with a 0.1 M buffer (pH 3.0), the activity of the biosorbent is regenerated and remained comparable for more than 200 cycles. Scanning electron microscopy reveals a successful immobilization of biomass on eggshells powder and precipitation of Cr (VI) on the bacterial cell surface. The proposed method proves highly sensitive and could be suitable for the determination of Cr (VI) at an ultra-trace level.


Introduction
Uncontrolled industrial discharge of toxic metals is continuously contaminating the air and aquatic environment. These toxic metals are non-biodegradable and possess an unfavorable tendency to accumulate in biological systems. Consequently, chemists and environmental engineers are actively engaged in designing simple, sensitive, low cost and eco-friendly processes to remove these toxic metals from waste waters. Different analytical instrumental techniques such as UV-Vis spectrophotometry, high performance liquid chromatography-flame atomic absorption spectrometry (HPLC-FAAS), electro-thermal atomic absorption spectrometry (ETAAS), and HPLC-inductively couple plasma mass spectrometry (HPLC-ICP-MS) have been exploited for the determination of toxic metals in environmental samples including wastewater, drinking water and soils [1]. Trace element determination is analytically challenging at low concentrations of the trace elements and also due to the potential interference of the sample matrix. A common technique to address the challenges is to pre-concentrate the trace elements prior to detection. A variety of sample pre-concentration methods including co-precipitation, liquid-phase extraction, HPLC and solid-phase extraction have been employed for the trace/ultra-trace level determination of toxic metals [2]. Solid-phase extraction is a preferred and the most widely used method, as it works even in the presence of interferences and is also safe from usage of toxic organic solvents. In batch methods these techniques face some limitations such as large reagent consumption, accumulation of waste sludge, being time consuming, loss of results reproducibility, risk of contamination of analyte [3]. These limitations can be restrained by the incorporation of solid-phase extraction technique in flow injection (FI) system.
Various approaches have been made to exploit lowcost agriculture and industrial wastes as sorbents for the removal of toxic metals [4][5][6][7][8][9][10][11][12][13][14]. In the last few years, biosorption/bioaccumulation processes have been studied extensively using microbial biomass as biosorbents for the removal of toxic metals [15][16][17][18][19][20][21][22]. Comparing to other adsorbents, microbial biomass (microalgae, fungi, bacteria) outperformed because of their strong affinity with the metallic species due to the presence of various functional groups on the microbial cell wall [23,24], such as phosphates, carboxyl, hydroxyl and amino groups [25]. The mechanism of the metal uptake by these microbes may be due to physicochemical sorption, complexation or bioaccumulation [26]. Both dead and living biomass could be applied for the removal of toxic metals, however the use of powdered biomass has some problems, such as difficulty in the separation and regeneration after use and small particle size makes it difficult to use in column applications [16,17,27,28]. The current state of the art of microbacteria-based sorbents for the preconcentration of metal ions at trace levels and various aspects of the biosorption technology comparing analytical figures of merit are reviewed [29][30][31]. Immobilization of the biomass on solid structures gives mechanical strength, rigidity to the biomass and also offers greater potential for its regeneration and reuse.
Cell immobilization has been initiated as an alternative for enzyme immobilization. Selection of the support material plays a crucial role in the process of immobilization and practical applications of microbial biomass [32]. Various support materials, such as calcium aliginate, alumina and nanomaterials like nanofibers are used in biomass immobilization by entrapment or physical adsorption methods [33][34][35][36][37][38]. Whereas controlled porosity glass (CPG) has been employed in biomass immobilization by covalent bonding method [35]. Immobilization by entrapment and physical adsorption has practical limitations such as inadequate transference of biomass in the inner part of support matrix, cell leakage, adverse effects on cell sustainability and abrasion of support matrix during use [39]. Covalent bonding is one of the most widely used methods for irreversible immobilization of biomass. Covalent bonding provides powerful links between the microbial cells and the carrier matrix thus preventing leakage of biomass, enhancing stability and viability of biomass for industrial and environmental applications and making them repeatedly useable. Therefore, a covalent bonding method is often the method of choice when other methods of immobilization are abortive. Different popular CPG preparations are commercially available as supports for immobilization, but are costly [40].
In this study, an effective and low-cost solid support of powdered eggshells is used for the immobilization of Pseudomonas aeruginosa. The mini glass column filled with immobilized bacterial biomass is incorporated in a flow injection system (CFS). Flow injection analysis is a versatile technique with multiple options of manifold design and the added benefits of minicolumns incorporated in the system, loaded with different materials for preconcentration and determination of various analytes. Chromium, an effluent of leather, tanning, paints, electroplating and textile industries, exists in two oxidation states: Cr (III) and Cr (VI). At low concentrations, Cr (III) is essential for living systems for the maintenance of various metabolic pathways whereas Cr (VI) is toxic even at low concentrations due to its carcinogenic effects [41][42][43]. In Pakistan, industrial waste waters contain Cr (VI) concentrations ranging from 0.002-35 mg L -1 whereas the Environment Protection Agency (EPA) has a drinking water standard of total chromium about 0.1 mg L -1 [44,45]. The purpose of this work is to develop a cost-effective, sensitive, efficient and green method for on-line enrichment and determination of Cr (VI) at ultra-trace levels. The analytical methodology described here employs eggshells powder for the first time as a solid support to immobilize microbial biomass for the on-line preconcentration and determination of Cr (VI). Various factors are optimized and working performance of the system to determine Cr (VI) was also studied. HNO 3 , potassium dichromate, 1,5-diphenyl carbazide (DPC) and H 2 SO 4 were purchased from Merck (Merck, Darmstadt, Germany). All the chemicals and reagents used in this study were of analytical grade. All glassware was soaked overnight in 10% HNO 3 , rinsed with distilled water and dried in an oven before use. 1000 ppm stock solution of Cr (VI) was prepared by dissolving 0.707 g of K 2 Cr 2 O 7 in 250 mL of distilled deionized water and working solutions were prepared in the range of 10-100 mg L -1 (ppm) and 10-100 µg L -1 (ppb) immediately before use by a stepwise dilution of stock solution in buffer solution of pH 3. A DPC solution was prepared by first dissolving 250 mg of DPC in 10 mL of ethanol with constant stirring using a magnetic stirrer and then diluted up to 100 mL with sulphuric acid (0.2 mol L -1 ).

Treatment of eggshells
Chicken eggshells were collected from the local market of Muzaffarabad, Pakistan and broken into pieces, followed by washing with acetone and being dried in an oven at 60°C. Washed pieces were then ground to powder, passed through sieves of 100 mesh and stored until further use.

Immobilization of Pseudomonas aeruginosa on eggshells powder
The microbiology laboratory of Zoology Department, University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan provided the pure culture of Pseudomonas aeruginosa. The bacterial strain was isolated from soil samples and identified using Gram's staining and biochemical tests. Pseudomonas aeruginosa (DSMZ 6195) was grown in a nutrient broth medium (Oxide: CM1) and incubated in a rotary shaker for 24 h at 37°C. The biomass was collected by centrifugation of culture mixture and the collected biomass was thoroughly washed with distilled water to remove the residual growth medium. The procedure was initiated with 10 mg of dried microbial biomass immobilized on 0.5 g of eggshell powder and the immobilized material was stored and only 100 mg was packed in column for flow injection process.
Immobilization of biomass was carried out using the method described by Maquleria et al. [35] with slight modification, here we used powdered eggshells as solid support instead of controlled porosity glass (CPG). First the bacterial solution was prepared by weighing 20 mg of sample of Pseudomonas aeruginosa into a small beaker (25 mL). After that, NaOH solution (0.5 g of NaOH pellets in 25 mL of water) was added with stirring to the beaker containing the bacteria and the pH of mixture was carefully adjusted to pH 7.0 with 1 M HCl (as in basic media, turbidity was observed). Then 10 mL of this bacterial solution was diluted to 25 mL with a phosphate buffer (pH 7) and ground eggshells (0.5 g 100 mesh size) were added into 15 mL of bacterial solution and stirred slowly for 15 mins with the addition of 25% glutraldehyde to bring the concentration to 2.0%. The mixture was stored at 4°C till further use. The successful immobilization of the microbes was observed with a scanning electron microscope (SEM) and infrared (IR) spectral analysis.

Flow injection manifold and procedure
A spectrophotometer (model UV 1800 Shimadzu, Kyoto, Japan) equipped with flow-through cell (30 µl volume and 10 mm path length) was used for absorbance measurement. A flow injection system consists of a reglo digital peristaltic pump (Ismatec, Wertheim, Germany) and six channel injection valve (Rheodyne RH 5020) with other basic components. Immobilized Pseudomonas aeruginosa (100 mg) packed in a glass minicolumn (5 cm length, 1.5 cm i.d) was incorporated in the system. The whole process consisted of three steps: preconcentration, washing, and elution. During preconcentration, 5 mL standard Cr (VI) solution was passed through the minicolumn using the peristaltic pump at the flow rate of 0.8 mL min -1 for 5 min. In order to remove the unadsorbed Cr (VI) from the column as well as the retained Cr (VI) from the tubes, 2 min washing was done with buffer (Phosphate-citrate), which reduces the risk of interference from unabsorbed Cr (VI) at the elution step. After washing, eluent was injected by the first injection valve followed by the injection of DPC from the second injection valve. The eluted Cr (VI) was mixed with DPC at mixing point and the purple-red colored complex formed, which was detected spectrophotometrically at 545 nm. The overall setup is presented in scheme 1 and the three-step flow injection on-line operation is presented in Figure 1. In order to establish the best chemical and flow conditions for flow injection on-line preconcentration and determination of Cr (VI) with good sensitivity and precision, various parameters such as pH (1-6), flow rate (0.7-1.6 mL min -1 ) and sample volume of Cr (VI) solution (5-20 mL) were optimized. Further, it is known that the reuse and recycling of the biosorbent for successive removal of metal ions from aqueous medium contributes to the economic feasibility of the biosorption process. A necessary factor is that the desorbing agent used for regeneration of the biomass should not damage the biosorbent. So, keeping this in mind, for regeneration of biomass, the effect of the type of reagents (NaCl, KNO 3 , HNO 3 and HCl) was studied and concentrations of the selected reagents in the range of 0.05-8 mol L -1 were also optimized. The optimized parameters and selected conditions for determination, preconcentration and regeneration of Cr (VI) are given in Table 1.
The percentage recovery (R, %) was calculated by the equation given below:

Immobilization of Pseudomonas aeruginosa on eggshells powder
In this study it was interesting to use eggshells for the immobilization of bacterial biomass instead of using any expensive solid support like CPG, which has been used for the immobilization of algal biomass [35]. In chicken eggshells, calcium carbonate crystals are stabilized by the Ovocleidin-17 (OC-17) protein matrix and without the protein crystal structure it would be too brittle to keep in original form [46]. Glutraldehyde carrying carbonyl bifunctional group is used in most of the immobilization procedures as a covalent cross-linking agent. Reaction chemistry for the immobilization of bacterial biomass on eggshells is given in scheme 2, which shows the linkage of carbonyl groups of glutraldehyde with -NH 2 of protein matrix of eggshell at one end and -NH 2 of bacterial cell wall at the other end. Cross-linking between solid support and biomass through glutraldehyde makes maximum surface area of biomass available for the interaction of metal ions on cell surface. Initial treatment of eggshells with acetone lessens the adhesion between egg membrane and eggshell and thus detaches the membrane from the shell and curtails the role of egg membrane in immobilization or biosorption procedures.

Effect of pH on biosorption and preconcentration of Cr (VI)
The pH is a very important factor and plays a vital role in the biosorption of a specific metal ion to the adsorbent surface. The effect of pH on preconcentration of Cr (VI) in a biosorption column is studied in the range of pH 1-6. Results presented in Figure 2A show that the percent recovery of Cr (VI) has a maximum at pH 3. The metal biosorption depends on the presence of protonated or unprotonated functional groups on the surface of the microbial cell wall and ionic state of the metal ions in solutions, which is correlated by the pH of the solution. Pseudomonas aeruginosa is a gram-negative bacterium and its cell wall is composed of peptidoglycan backbone, rich in phosphate, carboxylate and amino groups. Functional groups containing nitrogen could be quaternized at pH ≤ 4 and the positive charges would magnetize the anions and the isoelectric point (pI) for gram-negative bacteria lies at low pH [47]. The possible explanation of high Cr (VI) adsorption at low pH could be due to the fact that when pH is low then the total charge on cells surface would be converted into positive and the Cr (VI) which is in anionic form binds electrostatically with microbial cells [48]. At acidic pH, negatively charged chromium species (chromate/dichromate) bind through electrostatic attraction to positively charged nitrogen containing functional groups on the surface of the bacterial cell wall. The possible explanation of high Cr (VI) preconcentration at pH 3 could be due to the fact that at this pH the total charge on cells surface would be converted into positive and the Cr (VI) which is in anionic form binds electrostatically with microbial cells. Further decrease in pH has a damaging effect on eggshell powder. A sharp decrease in % recovery is observed at pH ≤ 3, which is due to the loss of microbial biomass from the damaged supporting material. Increase in pH from 4-6 has a decreased number of protonated functional groups, consequently a decrease in % recovery is observed.

Effect of sample flow rate on biosorption and preconcentration of Cr (VI)
Flow rate also plays an important role in controlling the sensitivity and sampling frequency in the flow injection system. The increase in flow rate increases sampling frequency but may decrease sensitivity. High flow rate also could exert back pressure, which may cause leakage or rupture of connections. The effect of flow rate on the preconcentration and determination of Cr (VI) is performed in the range of 0.7-1.6 mL min -1 . The results show ( Figure  2B) that as the flow rate is increased from 0.7 to 0.8 mL min -1 , there is an increase in Cr (VI) adsorption and after that a decrease in Cr (VI) adsorption is observed when the flow rate is increased from 1 to 1.6 mL min -1 . At the higher flow rate, back pressure and leakage at connection points is observed which may be the reason for low adsorption of Cr (VI). Although lower flow rate decreases sample throughput rate but for stability in flow injection system it is often a better choice. The flow rate of 0.8 mL min -1 is selected for further study.

Effect of sample volume on biosorption and preconcentration of Cr (VI)
The volume of the loaded metal ion solution is an important factor to be optimized in order to identify the maximum saturation potential of the adsorbant. In the present study, 5-20 mL of the sample is loaded in order to determine maximum uptake of the analyte. Results of the study depicted in Figure 2C reveals that there is a small increase in % recovery with an increase in sample volume with no further increase after 15 mL of loaded volume. This may be attributed to the fact that with an increase in loaded sample volume, the sites available for sorption become fewer as compared to the moles of analyte present and hence preconcentration of the analyte is strongly dependent upon the volume of the loaded sample. Small changes in % recovery with an increase in sample volume may also be linked to the small size of biosorption material in packed column in flow injection system. Although there is a steady increase in % recovery with an increase in loaded volume (5-15 mL) yet 5 mL of the volume is selected for further studies to increase the sample throughput rate with less effect on the sensitivity of the method.

Effect of various reagents on regeneration of biomass
Appropriate selection of eluents for a successful desorption process depends on the type of biosorbent and mechanism of biosorption. It is important that eluent must be non-damaging to the biomass, cheap, effective and environment friendly. Although some chemical agents performed very well, like acids and alkalies in the case of desorption of cations and anions, they are highly damaging for microbial cell surface [30]. Most of the previous studies were aimed at the binding ability of biomass but less attention has been focused on the regeneration of biomass. This study has vitally focused on the probability of regeneration of biomass to make the process applicable for practical applications. For this purpose, various eluents (NaCl, KNO 3 , HNO 3 and HCl) in the concentration range (0.05-8 mol L -1 ) are studied. Results of the study presented in Figure 3 clearly demonstrate that NaCl has significant potential for maximum desorption of the analyte from the biosorption column and KNO 3 also showed good desorption from the column. Significant desorption potential of the salts with increase in ionic strength is due to destabilization of electrostatic interactions between negatively charged chromium species (chromate/dichromate) and positively charged nitrogen containing functional groups on the surface of the bacterial cell wall.
While HCl and HNO 3 show weaker action and also badly damage the efficiency of biosorption column which may be due to the damaging effects of the acids on the microbial cell surface and the solid support (eggshells). Concentration and volume of the eluents also influence the elution process and would be important for complete desorption of the analyte, because a too-low eluent concentration would elute the analyte incompletely from preconcentration column and on the other hand, too high eluent concentration would waste the reagent. As in this study, a minimal fixed volume (100 µL) is used for eluent. The optimum concentration of 7 M for NaCl is selected for complete desorption of the analyte, which is confirmed by successive injections of the eluent and chromogenic reagent.

Interference studies
In fact, biosorption is a proven technique for having the potential of adsorption of metal ions. However, its application on real water samples is a great concern. Interference in real samples may be anionic or cationic species, which have an affinity for biosorbents and compete with the species of interest. Interference may also be offered from other metal ions, which can make a complex with DPC. Although DPC is very sensitive and almost specific for Cr (VI), there are several chemical species that may interfere either by complexing by DPC or by reducing Cr (VI) into Cr (III). In this study interference of some anions like; NO 3 -, PO 4 -, SO 4 and CO 3 at a concentration range of (10-100 mg L -1 ) are studied in the analysis of sample solution containing 10 mg L -1 of Cr (VI). Percentage interference of studied anionic species in the studied concentration range is found to be insignificant.

Examination of immobilized biomass before and after preconcentration
Eggshell, besides being widely available at almost no cost, also has good mechanical strength and resistance to microbial growth and are reported as good carriers for the immobilization of certain enzymes [49][50][51]. In current research, immobilization of biomass and biosorption of Cr (VI) is confirmed by scanning electron microscopy (SEM). The SEM images of eggshells powder, P. aeruginosa, immobilized P. aeruginosa onto eggshells powder and adsorbed Cr (VI) on immobilized P. aeruginosa onto eggshells are shown in Figure 4. The SEM images confirm the immobilization of P. aeruginosa onto eggshells ( Figure  4B) and the adsorption of Cr (VI) on immobilized P. aeruginosa ( Figure 4C). The Figure 4C clearly shows that Cr (VI) has covered the complete surface of the immobilized P. aeruginosa.
Infrared spectral studies are also carried out to observe the immobilization of biomass and biosorption of Cr (VI). The Figure S1A shows the IR spectra of the 100 mesh eggshells powder and Figure S1B shows the static biomass, P. aeruginosa immobilized on eggshells powder. In both Figures S1A and S1B, the IR spectra reveal the presence of certain functional groups on the surface of eggshells powder before and after immobilization. It can be observed that many peaks are shifted, some are retained, and some have disappeared while new peaks are also identified after immobilization of P. aeruginosa. A peak at 1423 cm -1 appears in Figure S1B and the peaks in the range of 3155-3542 cm -1 disappear in Figure S1C. The peaks at 3442 and 3220 cm -1 were retained in Figure S1B, supporting successful immobilization of biomass. These peaks might be due to -NH 2 asymmetric stretching mode of amines showed the overlapping of hydroxyl and amines stretching on the surface of bacterial cell [52]. IR spectral analysis is also carried out after loading of Cr (VI) ( Figure  S1C). The infrared spectra of the Cr (VI) loaded biomass are almost similar to that of the raw biomass. There is decrease and shift of peak at 890 cm -1 in the Cr (VI) exposed biomass ( Figure S1C) compared to unexposed biomass ( Figure S1B) indicating -CH=CH of trans-di-substituted alkenes out of plane deformation showing the strong interaction of Cr (VI) with bacterial cell wall [21]. Further SEM images also confirm the adsorption of Cr (VI) and Figure 4C clearly shows that Cr (VI) covered the complete surface of immobilized biomass.

Analytical and working performance of the system
After optimization and characterization, the performance of the system in the sense of calibration range and detection limit is studied. Under the optimized conditions, various concentrations Cr (VI) are injected into the flow system. The calibration curve is linear over the concentration range of 10-100 µg L -1 with a detection limit of 6.25 µg L -1 . Improvement in enhancement factors (EF) and sensitivity of the system are determined by taking ratio of slopes of the curves (0.0034/0.0006) and of detection limits (5.30 ppm/6.25 ppb), obtained from linear calibration curves plotted before and after preconcentration. Results of the study given in Table 2 show that both enhancement factors and sensitivity are remarkably enhanced after preconcentration. Further the column has retained its activity for more than 200 cycles. A comparison of analytical characteristics of Cr on bacteria already reported in literature is presented in Table 3. In our future study, we plan to work on the selectivity of this method towards different cations and also this established method will be extended for the determination of other toxic metals.

Conclusions
A simple analytical procedure with improved physicochemical features as well as enhanced biosorption characteristics has been designed. The methodology described here employs eggshells powder for the first time as a solid support for the immobilization of microbial biomass for the on-line retention of Cr (VI) followed by its elution and spectrophotometric detection. Under optimized conditions, the method has a detection limit of 6.25 ppb, with 848 times increase in sensitivity. The results have revealed excellent reproducibility for Cr (VI). For the better economic value of the biosorption procedure, repeated reuse of the biosorbent with minimum loss of efficiency by desorption of the metal pollutant as well as regeneration of the biosorbent is successfully achieved. The metal burdened biomass packed in a minicolumn retained its activity for more than 200 cycles by using salt solution as eluent. In addition, the proposed method is simple, economical, fast, highly sensitive and environment friendly which can be used complimentarily with other traditional metal removal technologies.