Bioadsorption of Cr (VI) in Aqueous Solutions by $Pseudomonas \,\, Koreensis$ Immobilized in Alginate Beads

The increase in industrialization and the negligence in the use of natural resources contribute to the release of heavy metals into aquifers. These elements represent a threat to health and the environment due to their toxicity, cumulative properties, persistence and immutable quality [1].

In recent years, the availability of water has decreased; in addition, contamination by heavy metals is a serious problem since these can remain a long time in water and in small quantities can cause damage to health. Chromium is considered one of the most toxic heavy metals [2]. Its main risks include carcinogenic potential [3], causing damage to the erythrocyte membranes [4], generating oxidative stress, causing DNA damage, cellular apoptosis and altered gene expression [4].

Due to the increase in water pollution, as well as the imbalance of aquatic ecosystems, more attention has been given to ecological measures, since one of the current challenges is to obtain safe and economical drinking water [1].

In this context, bioremediation is a promising alternative for the treatment of water with a low concentration of metals [5]. Among the advantages of bioremediation is that it is easy to implement. it is free of secondary contamination and have low costs [6]. Pseudomonas is one of the most used bacteria for bioremediation since it is adaptable to different environments. This genus is of great importance in wastewater treatment processes due to its high capacity to biodegrade many organic compounds [3]. Some species of Pseudomonas have been studied for chromium biosorption (P. putida, P. mendocina and P. aeruginosa) [7, 8]. P. aeruginosa has shown a high degree of multiple resistance to heavy metals [7].

On the other side, it is known that commonly in polluted areas by effluents from mining occur heavy metal tolerant microorganisms. In Mexico, the largest gold mine in America is located in Mazapil, Zacatecas [9]. Then, it is feasible to find bacteria adapted to this environment which could be useful to remove heavy metals from water, such as chromium.

Some of the disadvantages of the use of microbial biomass in suspension include its particle size, its low mechanical resistance and the complexity in separating the biomass from the effluent. For this reason, a wide variety of inert materials are used as support for microorganisms which allow their immobilization through a biofilm formation [10]. Biofilms improve the adsorption properties of the support, inducing greater resistance to metals and allow an easy separation of biomass from the substrate [11]. The materials evaluated for the immobilization of microbial biomass are agar, cellulose, alginates, silicate, among others [12-15]. Alginate stands out for its properties as biocompatibility and hydrophilicity, as well as being considered a non-toxic substance [16].

The goal of this work was to isolate a chromium tolerant microorganism from a natural watercourse near to a mining area. The isolated microorganism was immobilized in alginate beads and its ability in Cr removal from water solution was evaluated.

The water samples were taken from a watercourse (Arroyo Grande) and from a spring (Las Goteras) located in Mazapil, Zacatecas, Mexico ($23^{\circ} 41^{\prime}, 25^{\circ} 04^{\prime} / 101^{\circ} 11^{\prime}$ and $102^{\circ} 41^{\prime}$). The town is near to deposits of zinc, gold, silver, copper, mercury, phosphorite, limestone, lulita, calcite, onyx and marble. Water samples were kept at $4{ }^{\circ} \mathrm{C}$ in a dark environment and all analyses were performed within 24 h , according to the Standard Methods for Examination of Water and Wastewater [17]. The identification of metal ions was performed by using a Thermo Model iCE 3000 atomic absorption spectrometer. Water samples were filtered, digested with $\mathrm{HNO}_3$, and analysed directly to determine metals concentration, according to the standard NMX-AA-051-SCFI-2001 [18].

For bacteria isolation, a serial dilution from a saline solution of $0.85 \%(10-1$ to $10-4)$ was conducted on the collected samples. After that, the microorganisms were cultured by duplicate in Nutrient agar, EMB, MacConkey and Mannitol salt agar. Plates were incubated at $30-35^{\circ} \mathrm{C}$ for 24 h and then microorganism count was performed. Dominant colonies were replanted in a nutrient agar supplemented with heavy metals solutions; they were incubated at $30-3^{\circ} \mathrm{C}$ until detect bacterial growth. For the molecular identification of bacteria, five isolates were chosen among those with the highest metal tolerance. The molecular identification was carried out by 16 S rDNA sequencing.

Alginate beads were elaborated by coprecipitation following the methodology reported by Calero et al. [19]. Briefly, an alginate solution ($3 \mathrm{~g} / 200 \mathrm{~mL}$) was placed in a separation funnel (which was previously conditioned with a micropipette nozzle at the end of the tip of the funnel) and a drip was started on a calcium chloride solution ($3 \mathrm{~g} / 200 \mathrm{~mL}$), in constant magnetic stirring. Finally, the beads were washed 3 times with distilled water.

For biofilm formation, 50 mL of Cetrimide broth (17 g Cetrimide agar / L) previously inoculated with $P. koreensis$ (24 h) and 5 g of calcium alginate beads were mixed. The mixture was incubated at $30^{\circ} \mathrm{C}$ with orbital shaking at 150 rpm for 24 h . The formation of the microbial biofilm was confirmed by Scanning Electron Microscopy (SEM): the samples were prepared with a fixative of $2 \%$ formaldehyde and $70 \%$ ethanol for 18 h, they were subsequently dehydrated in gradients of ascending ethanol: $70 \%, 80 \%$ and $95 \%$.

The Cr (VI) bioadsorption tests were carried out in 250 mL reactors. The pH of the solution was adjusted to 6.6 with a phosphate buffer $(50 \mathrm{Mm})$, and kept in soft agitation: 100 rpm at $30^{\circ} \mathrm{C}$. The contaminant concentration of Cr (VI) was 10 ppm, which was prepared from a stock solution of $1000 \mathrm{ppm}\left(2828 \mathrm{~g}\right.$ of $\left.\mathrm{K}_2 \mathrm{Cr}_2 \mathrm{O}_7 \mathrm{~L}^{-1}\right)$ [2]. The concentrations of bioadsorbent were 15, 25 and 40 g, and the reaction volume was 150 mL. The final concentration of metal was determined by atomic absorption spectrometry (AAS) (ICE 3000 SERIES AA SPECTOMETER). Samples were taken at 0, 4, 8, 24 and 32 h [2, 20-22]. The experiments were carried out by triplicate.

From bacterial isolation of the collected samples, a multi-resistant bacterium to $\mathrm{Zn}, \mathrm{Ag}, \mathrm{Hg}$ and Co with MIC of 2500, 2500, 85, 105 ppm, respectively, was detected. In addition, this bacterium exhibited intermediate and susceptible responses to Cr. The molecular identification of this bacterium by 16 S rDNA indicated that it is a Pseudomonas koreensis.

Pseudomonas was the most tolerant to all metals tested, showing an increase in its growth compared to the culture without heavy metals. This was probably due to synergetic effects in the enzymatic catalysis; as the metal concentration increase its toxicity, the stimulating effect decreases, and the system is eventually inhibited [23]. The resistance of this microorganism to metals is probably related to the incorporation of those elements to cytoplasm, once they bind to specific metallothioneins [24, 25]. Since P. koreensis is a Gram-negative bacterium, it has a higher resistance to the toxic effects of heavy metals due to its cellular wall, which hinders their incorporation into inner cells [24]. These significant differences in the resistance to heavy metals could be related to variances in the isolation site and to adaptation mechanisms developed by bacteria. Other studies reveal a relationship in tolerance to Zn, Cr, and Cu because they are used as micronutrients for different metabolic pathways and tend to bioaccumulate when they exceed certain levels close to MIC [26, 27]; this could explain the increase growth for all bacteria in presence of these metals.

Rounded, soft and clear spheres of alginate were obtained by using a nozzle of 1 to 10 mL (blue tip). 65 g of alginate beads were obtained (wet weight); the average diameter of these beads was 3 mm. Figure 1 shows representative sample of the obtained alginate beads.

The viability of the system is verified by microscopy. A sample was taken at the end of the bacteria growth experiment and a gram stain was performed. As shown in Figure 2, gramnegative bacillus was detected; this bacillus is characteristic of Pseudomonas.

Regarding the adhesion of bacteria to the support, representative samples of alginate beads were taken from the immobilization system at 8,12 and 24 h to be analyzed. For comparative purposes, a sample of virgin beads was also evaluated. The obtained results are presented in Figures 3-6.

The micrograph corresponding to the alginate beads sampled at 8 h shows the adhesion of P. koreensis. It is possible to observe microorganism colonies randomly dispersed (Figure 3). The analysis of the micrographs also reveals the presence of cellular aggregates, hollows and biomass embedded in the alginate matrix, of approximately $1 \mu \mathrm{~m}$ in length, which agrees with the morphology of P. koreensis. This was confirmed by means of EDS, since there are elements characteristic of the prokaryotic bacterium wall (F, N, K). None of the aforementioned characteristics were observed in the virgin beads (Figure 6).

The observed morphology is similar to that reported by Mangwani; the authors studied the immobilization of P. mendocina on glass. In their study, using SEM they verified the presence of a biofilm conformed by bacillar aggregates [28].

Data obtained from the experiments were analyzed using the software STATISTICA 7.0, a factor analysis with $95 \%$ confidence was considered. Table 1 presents the results of the analysis of variance with the associated variability of the Time and Bioadsorbent dose factors and their interactions. The effect of each factor is defined as the response produced by a change in the level of the factor. All the terms were statistically significant since they have $p$-values lower than 0.05 ($95 \%$ confidence level).

It is observed that time and bioadsorbent concentration, as well as their interactions, influence the bioadsorption of Cr (VI). Since there are significant differences between the treatments and the reference, the results are analyzed by Fisher's test to determine which of the treatments are different from each other. Fisher's test is useful to compare two categorical variables and find if there are significant differences in each of the treatments.

The Fisher test was carried out in the software STATISTICA 7.0 (Table 2). It is shown that the best treatments correspond to 32,48 and 56 h , with a bioadsorbent concentration of 40 g. No significant differences were found between them considering a $95 \%$ confidence. Then, it is determined that the best treatment is 40 g of bioadsorbent at 32 h, since there are no significant differences if the time is increased.

The Cr adsorption kinetics of $P. koreensis$ is presented in Figure 7. The parameters used were pH 6.6, temperature $30^{\circ} \mathrm{C}$, initial concentration of Cr (VI) 10 ppm and biosorbent dosage $120 \mathrm{~g} / \mathrm{L}$ (wet weight). An adsorption efficiency of $97 \%$ is obtained at 32 h. This percentage of adsorption agrees the reported for Pb (II) adsorption using Sargassum filipendula [29]; in that work a removal efficiency of $96 \%$ was reached. In another study, the removal percentage of Cu (II) was $80 \%$ at four days, considering an initial concentration of 200 ppm, using as adsorbent a microalgae/bacteria system [30].

Immobilization of P. koreensis was corroborated by SEM and EDS analysis. A significant difference was observed regarding the adsorbent concentration and time. The best result was $97 \% \mathrm{Cr}$ (VI) removal at 32 h , which correspond to 40 g of adsorbent. P. koreensis seems to be an adequate biosorbent material of heavy metals; this is a possible alternative to minimize the problem of water pollution.