A statistical approach of zinc remediation using acidophilic bacterium via an integrated approach of bioleaching enhanced electrokinetic remediation (BEER) technology

Chemosphere 207 (2018) 753e763

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A statistical approach of zinc remediation using acidophilic bacterium via an integrated approach of bioleaching enhanced electrokinetic remediation (BEER) technology Adikesavan Selvi**, Rajasekar Aruliah* Environmental Molecular Microbiology Research Laboratory, Department of Biotechnology, Thiruvalluvar University, Serkkadu, Vellore, 632115, Tamilnadu, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


An acidophilic bacterium was isolated from tannery effluent contaminated sludge.
Bioleaching and Electrokinetic process parameters were optimised using a statistical model.
An integrated method of Bioleaching Enhanced Electrokinetic remediation was demonstrated for zinc removal in real soil.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 March 2018 Received in revised form 17 May 2018 Accepted 24 May 2018 Available online 25 May 2018

The aim of the present study was to isolate an indigenous acidophilic bacterium from tannery effluent contaminated sludge (TECS) sample and evaluate its potentiality towards the removal of zinc using an integrated approach of bioleaching enhanced electrokinetic remediation (BEER) technology in zinc spiked soil at an initial concentration of 1000 mg/kg. The isolated acidophilic bacterium was characterized by biochemical and 16S rRNA molecular identification and was named as Serratia marcescens SMAR1 bearing an accession no. MG742410 in NCBI database. The effect of pH and inoculum dosage of SMAR 1 strain showed an optimal growth at pH 5.0 and 4% (v/v) respectively. Based on these experimental data, a statistical analysis was done using Design Expert computer software, v11 to study the interaction between the process parameters with respect to zinc reduction as an output response. Electrokinetic experiments were conducted in a customised EK cell under optimised process conditions, employing titanium electrodes. Experiments for zinc removal were demonstrated for bioleaching, electrokinetic (EK) and BEER technology. On comparing, the integrated process was found to evidence as an excellent metal remediation option with a maximum zinc removal of 93.08% in 72 h than plain bioleaching (72.86%) and EK (56.67%) in 96 h. This is the first report of zinc removal in a short period of time using Serratia marcescens. It is therefore concluded that the BEER approach can be regarded as an effective technology in cleaning up the metal contaminated environment with an easy recovery and reuse option within short period of time. © 2018 Elsevier Ltd. All rights reserved.

Handling Editor: X. Cao Keywords: Acidophilic Bioleaching Electrokinetic Response surface methodology Zinc

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (A. Selvi), [email protected], [email protected] (R. Aruliah). https://doi.org/10.1016/j.chemosphere.2018.05.144 0045-6535/© 2018 Elsevier Ltd. All rights reserved.

1. Introduction Heavy

metals

constitute

a

group

of

highly

persistent

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environmental contaminants, due to which their effect on biotic forms are highly extreme. Their nature of resistant to degradation by natural means has led to bioaccumulation and biomagnification of heavy metals in the food chain (J€ arup, 2003; Rzymski et al., 2014). Though these metals exists as natural elements of the earth's crust, their existence in the soil could be greatly contributed to various sources of anthropogenic activities like, improper disposal of electronic waste, industrial discharges, improper domestic waste management practices, unorganised dumping, use of fertilizers, transportation, etc. (Herawati et al., 2000; Goyer, 2001; He et al., 2005; Szyczewski et al., 2009). As a result of these, a drastic alteration in the biochemical balance and geochemical cycles have been reported (Bradl, 2002). In general, few of the heavy metals namely, zinc (Zn), copper (Cu), manganese (Mn), nickel (Ni) etc., are grouped as essential micronutrients, that are needed at very low concentration for certain biological metabolism in living organisms (WHO/FAO/IAEA, 1996; Wang et al., 2014). On the other hand, heavy metals like, mercury (Hg), lead (Pb), cadmium (Cd) and Arsenic (As) etc., don't have any specific metabolic role and hence are categorised as obligatory toxic (Rzymski et al., 2015). However, both the group of heavy metal's uptake could be highly toxic at higher concentrations causing serious health effects like tubular damage in kidneys, lung damage (Mandel et al., 1995; Hotz et al., 1999), coronary heart disease (Salonen et al., 1995), lung cancer, stomach cancer and gliomas (IARC, 1993; Steenland and Boffetta, 2000; WHO, 2001) brain damage, psychological and neurological symptoms, such as shivering, personality changes, anxiety, restlessness, sleep distur€rup, 2003). bances and depression (Ja Over the past, various heavy metal removal/recovery methods like, chlorination (Fraissler et al., 2009), chemical extraction (Marinos et al., 2007), electrokinetics (Violetta and Sergio, 2009), ion exchange (De Villiers et al., 1995), membrane separation (Chaudry et al., 1997), and bioleaching methods (Pathak et al., 2009) from soil/sludge have been reported by many researchers. Owing to advantages such as, cost-effectiveness, efficiency and less energy consumption, biological methods are much preferred than any other methods (Fang et al., 2014; Chi and Gao, 2015). Bioleaching is one such effective approach that is commonly employed to recover/ remove the heavy metals from soil/sludge. In this method, the metals are dissolved under the acidic condition by the action of acidophilic microorganisms (Pathak et al., 2009; Govarthanan et al., 2014). Unfortunately, factors like, microbial acclimatization time and biodegradable efficiency of the indigenous microorganisms greatly limits its application, if the process is employed alone (Mrozik and Piotrowska-Seget, 2010). Likewise, electrokinetic remediation (ER) method is also equally preferred as biological methods because of its simple operation, effectiveness, and no subsequent pollution for the removal of heavy metals from soil/ sludge (Zhou et al., 2004; Deng et al., 2009; Violetta and Sergio, 2009; Ma et al., 2010). Similar to bioleaching, ER method has also certain restrictions like, bioavailability of the heavy metal and mass transfer between the electrode and pollutants (Lohner and Tiehm, 2009; Simoni et al., 2001). But these restrictions can be overcome by combining bioleaching with electrokinetic (EK) remediation method, as this combination is believed to promote increased bioavailability of the pollutants, enhancement in biodegradation efficiency by generating oxidization and reduction zones, releasing of soil/sediment bound pollutant, improved nutrient transport, improved performance, and availability of terminal electron acceptors (Maini et al., 2000; Luo et al., 2005; Wick, 2009; Kim et al., 2010; Peng et al., 2011). In addition, the bioleaching process combined with electrokinetic technology is considered advantageous with an extended function of recycling the metals for other applications.

Since we are integrating two different approaches, the possible outcomes of the experiment are evaluated using RSM software by optimizing different variables (pH, inoculum dosage and contact time) chosen for the present study. This is done to reduce the time and the number of experimental trials by calculating the number of possibilities of variables and their interactions. Out of other models, Box-Behnken design (BBD) used in our study is considered to be one of the superior statistical designs which mathematically compute the significance of many variables in less number of experiments (Ojha and Das, 2018). Therefore, the present study is carried out to demonstrate the feasibility of heavy metal removal using an integrated approach of bioleaching and enhanced electrokinetic remediation (BEER) technology at lab scale, under optimised operative conditions on real soil spiked with zinc. In this study, we have chosen zinc (Zn) as a model pollutant because of its increased soil concentration due to numerous anthropogenic inputs, globally (Zhang et al., 2012; Cruz et al., 2014; Moreira et al., 2016; Lu et al., 2017). Moreover, Zn is considered to be one of the most mobile and potentially toxic heavy metals present in the environment (Mishra et al., 2008; Gondek and Mierzwa-Hersztek, 2016). Their non-volatilising nature and partial leaching were reported to cause acute and chronic effects in both aquatic and terrestrial biota (Chen et al., 2010; Milosavljevi c et al., 2011). Presence of Zn in soils, more than 0.6 mg/kg was reported to interfere with plant metabolism in absorbing other essential metals, like manganese and iron (WHO, 1996; Cameron, 1992). Hence, an integrated attempt demonstrated in our present study is expected to remediate polluted territory to pristine state. 2. Materials and methods 2.1. Chemicals and reagents Luria-Bertani (LB) agar, Tris Hcl, EDTA, Glucose, and Zinc sulphate were procured from Himedia Laboratories, India, Pvt. Ltd. Sulphuric acid (95e98%) and nitric acid (>90%) were purchased from Sigma-Aldrich, India, Pvt. Ltd. All the chemicals and acid solutions used in this study were of analytical grade and used without further purification. 2.2. Sample collection The tannery effluent contaminated sludge (TECS) was collected from a tannery industry located at Peranambut (12.9447 N, 78.8708 E), Vellore District, Tamilnadu, India. A sample size of 10 kg was collected in a clean plastic bucket provided with lid, using a clean mini-shovel. The samples were kept at 37  C until we reached laboratory from the collection site. The samples were stored at 4  C, until isolation. 2.3. Enrichment of acidophilic microorganisms The enrichment procedure was carried out following the protocol by Xiang et al. (1998) with slight modifications. Briefly, 50 g of the collected soil was added to 100 ml of distilled water and incubated at 30  C in a shaker incubator at 150 rpm for 7 days. 3 g/L of glucose was added as a sole carbon source. At the end of incubation, 10 ml of the soil suspension from the above culture flasks (test flasks) was transferred to five separate Erlenmeyer flasks containing 100 ml of sterile distilled water. The pH was adjusted to 2.0e6.0 using 1 N H2SO4. Sterile Erlenmeyer flasks containing sterile distilled water adjusted to the pH range of 2.0e6.0, served as a control. The set up was incubated at 30  C in a shaker incubator at 150 rpm for 3 days.

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2.4. Isolation of acidophilic bacteria

2.7. Effect of pH on growth of isolates

The enriched cultures grown in the above preparations were plated in Luria-Bertani (LB) agar containing glucose (3 g/L) by spread culture technique. The petridishes were incubated for 3 days at 30  C. The grown isolates were streaked on to fresh agar plates to obtain pure culture. The pure cultured isolate was stored in slants for future use.

The effect of pH on the growth of isolates was studied with respect to viability of the isolates at different pH ranging from 3.0 to 6.0. The isolate was cultured in LB broth containing glucose was maintained for 6 days at 30  C in a shaker incubator at 150 rpm. The pH of the LB medium was adjusted to the respective pH for each range (3.0e6.0). Appropriate control flasks were also maintained for each pH. The isolates were withdrawn at a regular interval of 24 h to check the growth by measuring the optical density (OD) at 640 nm. The grown isolates were subsequently sub-cultured in the same medium and stored at 4  C for future experimental applications. The isolates were also acclimatized to gradual increasing concentrations of zinc sulphate and stored in the refrigerator for future studies.

2.5. Biochemical and molecular identification of the isolate The bacterial isolate was grown on pH adjusted LB medium until late exponential phase. Then, the culture was harvested by centrifugation at 10000 rpm for 10 min. The procedure for extraction of high molecular weight genomic DNA from bacterial cells was done using high pure PCR template Preparation kit, Roche. The extracted DNA sample was dissolved in TE buffer of composition 10 mM Tris HCl and 1 mM EDTA (pH 8.0). This sample was used for amplification studies by polymerase chain reaction (PCR) employing universal primers as follows, forward primer 27F-50 AGAGTTTGATCCTGGCTCAG-30 and reverse primer 1492R-50 TACGGYTACCTTGTTACGACTT-30 . The PCR master mix of 25 mL volume consisted of double distilled water-18.3 ml, Taq DNA polymerase buffer (10x)-2.5 ml, dNTP mix (2 mM)-2.5 ml, forward primers (10 mM)-0.5 ml, reverse primers (10 mM)-0.5 ml, template DNA-0.5 ml, Taq DNA polymerase enzyme (5U/ml)-0.2 ml (Sambrook et al., 1989). PCR amplification was carried out for 35 cycles under following conditions of denaturation at 94  C for 45 s, annealing at 55  C for 45 s, extension at 72  C for 1 min and final extension at 72  C for 5 min. The amplicon comprised of partial sequences of 16S rRNA were purified by MACHEREY-NAGEL PCR clean up kit. The amplicons were subjected to Sanger di-deoxy DNA sequencing using the primers as follows, 518F-50 -CCAGCAGCCGCGGTAATACG30 and 800R-50 -TACCAGGGTATCTAATCC-30 . The sequences were determined using an automated DNA sequencing system (Model No. 3730XL, Applied Biosystems, USA). BLAST (Basic Local Alignment Search Tool) program, version 2.2.20 was used for similarity search from the NCBI (National Centre for Biotechnology Information) taxonomy and ribosomal database Project II-Release 10 (Altschul et al., 1990). Based on the similarity results, phylogenetic analysis was performed using CLUSTAL W, version 2.1 (DDBJ-DNA, Data Bank of Japan). A phylogenetic tree was constructed by neighbour-joining method using TREEVIEW software, version 1.6.6 and similitude percentages between sequences were analysed with 1000 random samples obtained from multiple alignment (Selvi et al., 2014; Rajasekar and Ting, 2014). The assembled 16S rRNA partial sequence of SMAR1 was deposited in GenBank and accession number, MG742410 was obtained for the isolate. 2.6. Soil spiking The collected TECS samples were air-dried at room temperature for 3 days and sieved through a 3 mm polyethylene sieve to remove the stones and other large debris. The protocol of soil spiking is followed according to procedure described elsewhere (Tripathi et al., 2016). From this, 500 g of TECS was weighed and packed in an autoclavable bag and autoclaved at 121  C for 15 min. The sterile soil was cooled to room temperature. To this, a fixed concentration of 1000 mg/kg of ZnSO4 (Zinc sulphate) was chosen as a form of zinc existence and added to the sterile soil (ATSDR, 1995; Minten et al., 2011). The soil was mixed well and left for 5 days at ambient temperature and later used for experiments. The spiked soil was studied by scanning electron microscopy (SEM, Model: JEOL - JSM6380, USA).

2.8. Effect of inoculum dosage The effect of inoculum dosage of the acclimatized bacterial strain SMAR1 was studied at different range (1e5% (v/v)) with respect to zinc reduction. The experiments were conducted in sterile Erlenmeyer flasks containing LB medium containing glucose along with added spiked TECS soil containing zinc in the form of ZnSO4 as described in the earlier section. The pH of the medium was maintained at an optimised range. Appropriate abiotic control flasks were also maintained along with the test flasks. All the flasks were incubated at 30  C at 150 rpm. The isolates were withdrawn at a regular intervals of 24 h to check the changes in the zinc concentration. Spectrophotometric zinc analysis was done by Dithiozone method (APHA, 1992) and the content was measured at 535 nm in a UVeVis spectrophotometer (Model, UV-1800, Shimadzu, Japan).

2.9. Statistical optimization of zinc remediation by response surface methodology (RSM) Statistical optimization of zinc remediation process parameters namely, pH (3.0e7.0), inoculum dosage (1e7%) and contact time (24e120 h) was studied by response surface methodology using Box-Behnken design (Selvi et al., 2015). The range of the parameters was fixed based on the preliminary experimental data. The data was evaluated using quadratic model in terms of single variable interaction at a time. The statistical analysis was performed using Design expert Version 11 (Stat-Ease Inc., Minneapolis, MN, USA) statistical software. Each factor was analysed at three different levels 1, 0 and þ 1. The design comprised of 17 runs. The percentage of zinc removal in soil was used as a dependent variable in the form of response, R1. The optimised parameters were evaluated using 3D contour plots, which influence the output response variable.

2.10. Demonstration of bioleaching process Bioleaching will be carried out in LB by growing the acidophilic strain, SMAR1 in 250 ml Erlenmeyer flasks containing 1000 mg/kg of ZnSO4. Fresh LB þ spiked TECS soil served as an abiotic control. The experiments were carried out under optimised pH and inoculum dosage. All the flasks were incubated at 30  C at 150 rpm. The samples were withdrawn at a regular interval of 24 h to check the changes in the metal concentration by Spectrophotometer method as described in earlier section (APHA, 1992).

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2.11. Integrated approach of bioleaching enhanced electrokinetic remediation (BEER) technology A customised electrokinetic remediation (ER) cell set up was used in our study. The set-up consisted of acrylic rectangular cells of dimension (22 cm  5 cm  7 cm3) divided into three compartments, in which the central one holding the ZnSO4 spiked TECS soil and 48 h grown SMAR1 strain at an optimised 4% (v/v) concentration. The other two side compartments were filled with plain distilled water, that served as working reservoir solutions of catholyte and anolyte respectively. Titanium electrodes of dimensions of 5.8 cm  9 cm  0.5 cm were used for the experiment. A pair of Whatman No.2 filter paper was placed in between the TECS bed and working reservoir solutions. A direct current (DC) power supply with an adjustable electric voltage (20 V) current was connected to the set up. The experiment was run with RSM optimised ER process parameters (pH, inoculum dosage and contact time). Then, the residual zinc (%) was calculated by collecting the soil samples from the central compartment at regular 24 h interval. The collected soil samples were air-dried at 20  C and subjected to acid digestion method (Edgell, 1988; USEPA, 2001). The residual zinc content in the soil was analysed by flame atomic absorption spectrometry, as recommended in standard protocols (Verlag Chemie, 1989; ISO, 1986). Experiments were terminated when there were negligible changes in the metal extract concentration.

respective SEM image are given in Fig. 1a and b. On analysing the colony morphology of the strain SMAR1, it looked like a circular, 0.5e0.8 mm sized, bright-orange coloured, slightly raised, semi-dry with regular edged, pigment producing bacterium (Fig. 1a). The morphology of the isolate is further studied by scanning electron microscopy (SEM), which showed a proper rod shaped, actively dividing live bacterium. The respective results are presented in Fig. 1b. The results of the biochemical tests are listed in Table 1. The standard biochemical tests showed rod-shaped, Gram negative, catalase, citrate and Voges-Proskauer positive organism. It also revealed the isolate to be motile, pigment producing and acid producing in the presence of glucose, maltose and sucrose carbon sources. The results of the morphology and biochemical results are found to confirm the bacterial isolate can be Serratia sp. (Holt et al., 1994). Molecular characterization of the bacterial isolate is done by partial sequences of 16S rRNA. The obtained sequences are compared with other bacterial gene sequences of NCBI using BLAST tool for pairwise identification. Based on this, a phylogenetic relatedness was derived in the form of phylogenetic tree by neighbour-joining method using TREEVIEW software and the results are presented in Fig. 2. According to the phylogenetic tree of the isolate, the bacterium is found to show its close relatedness to Serratia marcescens strain 1274 (Acc. No. CP019927.2). Therefore, the isolated bacterial strain is named as Serratia marcescens SMAR1.

3. Results and discussion 3.1. Isolation of acidophilic bacterium An indigenous acidophilic bacterium, designated as SMAR1 strain is isolated from tannery effluent contaminated sludge (TECS) discharge at Pernambut, Vellore Dist (Fig S1). The acidophilic bacterial isolation is done by the simple enrichment method followed by screening of the isolate as reported in previous studies (Baumgart, 2003). Enrichment step is believed to enhance rapid growth, good visible and identifiable colonies (Karkhane et al., 2010). 3.2. Morphological, biochemical and molecular characterization of the bacterial isolate The results of the isolate (pure culture) grown in LB plate and its

Table 1 Biochemical characterization of bacterial strain, SMAR1. Tests

Results

Shape Gram staining Capsule Catalase Citrate utilization Indole Motility Methyl Red (MR) Oxidase Pigment VP (Voges Proskauer) Acid from glucose Acid from maltose Acid from Sucrose Acid from Lactose

Rods Negative Negative Positive Positive Negative Positive Negative Negative Positive Positive Positive Positive Positive Negative

Fig. 1. Morphological characterization of the bacterial strain, SMAR1. (a) Plate colony morphology, and (b) SEM micrograph of the fixed bacterium.

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Fig. 2. A phylogenetic tree showing the genetic relatedness of Serratia marcescens, SMAR1 with closely related bacterial strains of NCBI library.

The obtained sequence has been deposited in GenBank library bearing an accession number, MG742410.

3.3. Effect of pH and inoculum dosage The pH value was found to be one of the critical factors that determine the growth of microbes, which in turn improves the removal efficiency of heavy metal in sludge (Mo et al., 2001). Thus, the effect of pH at different acidic range was studied and the results are presented in Fig. 3a. A maximum growth of Serratia marcescens SMAR1 at 600 nm is taken as a measure to determine the optimal pH range. Though the growth of the strain SMAR1 seem to be fairly

good in all acidic pH, a profound increase in the growth is noted at pH 5.0. According to earlier reports, Serratia marcescens was reported to exhibit a good growth rate at pH 5.0 (Slonczewski and John, 2009). This acidic pH range is believed to favour the metal extraction/removal from soil, thus by, detoxifying the heavy metal contaminated environments as reported in many previous studies (Mulligan et al., 2001; Lavalle et al., 2005; Suzuki et al., 2014). Unlike the extremely low pH, a near neutral pH of 6.0e7.0 can be established for extraction/removal of some metals, such as zinc, cadmium and arsenic (INECAR, 2000; Lenntech Water Treatment and Air Purification, 2004). Thus, the optimal pH range of our study to remove zinc seems to coordinate well with the previous

Fig. 3. Effect of process parameters (a) Effect of pH, and (b) Effect of Inoculum dosage.

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reports. Similarly, the effect of inoculum dosage of the acidophilic bacterial strain SMAR1 towards metal removal is also studied and the results are presented in Fig. 3b. The obtained results shows the lowest residual zinc percentage of 27.14 at 4% (v/v) of inoculum dosage. At the same time, there is no significant changes in the zinc concentration in case of abiotic control. This confirms the significant role of the acidophilic isolate, Serratia marcescens SMAR1 towards changes in the zinc concentration present in the spiked soil sample. As reported in earlier studies, an increased and maximum removal rate of Cr(VI) bioreduction was achieved at high inoculum size than compared to a low inoculum size (Ezaka, 2012; Sepehr et al., 2005). However, this is the first report on changes in zinc removal with respect to effect of inoculum dosage of Serratia marcescens SMAR1.

composite design (CCD) to study the effects of input variables towards removal of lead and nickel from sand as an output response was reported (Guaracho et al., 2009). In this study, RSM was employed to study the individual and interaction effects of three input variables with respect to the heavy metal removal. In an another study, RSM was used to design the electrokinetic reactor for vanadium recovery (Broska da Cruz Deniz et al., 2017) and field scale implementation of electrokinetic treatment of toxic metal in dredged sediments (Masi et al., 2017). Thus the results of the RSM software provided insights about the probable interaction between the chosen parameters to achieve an effective zinc removal from the real soil. It also has given options on implying the treatment option to field scale level in our future attempts.

3.4. Statistical optimization of zinc remediation by response surface methodology (RSM)

The electrokinetic remediation is considered as a promising remediation option with high efficiency, low cost and produces no subsequent pollution (Yuan and Weng, 2006; Apostolos et al., 2009). A customised electrokinetic remediation (ER) cell set up, used in the present study is shown in Fig. 5a. As the process parameters, such as pH, inoculum dosage and contact time were already optimised by RSM, these optimised results are implemented for bioleaching and integrated process of BEER technology. The comparative graph of both the processes is presented in Fig. 5b. The results of the bioleaching showed a maximal zinc reduction of 72.86% at end of 5 days of treatment. The obtained results are found to be well coinciding with the growth pattern of the acidophilic isolate, Serratia marcescens SMAR1, showing a maximum zinc removal from 2nd to 4th day, the exponential phase of the our isolate. As the day proceeded to 5e6th day, a steep decrease or slowdown in the bioleaching process was noted. This trend confirmed the active involvement of our isolate in bioleaching process at optimised process conditions. This was further confirmed by checking the viability of the bacterium by standard MPN count technique, which showed a good agreement with the growth trend of the strain SMAR 1. There are various reports on bioleaching involving Thiobacillus sp. and Acidothiobacillus sp. towards removal of heavy metals like Cu, Zn, Ni, Au removal at pH 4.0 (Tichy et al., 1993; Song et al., 2013; Nguyen et al., 2015). However, this is the first report on Serratia marcescens towards zinc removal from soil. Bioleaching processes, employing acidophilic bacterial isolates with acid producing capability has reported as an underlying mechanism to achieve an appreciable metal removal from various heavy metal polluted environments. On this context, microorganisms such as, bacteria and fungi involved in the production of low molecular weight metabolites, viz., pyruvic acid, gluconic acid, oxalic acid, citric acid, and succinic acid were reported to be effective bioleaching community (Rezza et al., 2001; Amiri et al., 2011). Likewise, our acidophilic isolate, Serratia marcescens SMAR1 is also found positive for pyruvic acid production, which is believed to play a role in bioleaching process. In a recent study by Xu and Feng (2016), reported on bioleaching with a maximum removal of 87.6% of Zn, 74.1% of Cu, 82.1% of Pb, and 97.8% of Cd. Therefore, bioleaching has always proved advantageous because of its environment-friendliness and low cost as reported by many researchers than the chemical leaching process (Deng et al., 2013; Yang et al., 2016). This is evident with our results too. Thus, the obtained results could be attributed to the active involvement of the indigenous acidophilic isolate, Serratia marcescens SMAR1. Similar to bioleaching, electrokinetic remediation is also considered to be one of the most efficient, viable and versatile processes in terms of removal of heavy metals from soils (Ottosen and Hansen, 1992; Yang et al., 2005; Jensen et al., 2007; Lima et al., 2011). However, these properties may sometimes get

Response surface methodology (RSM) is employed for optimizing the response, the output variable that is influenced by various independent factors, the input variables (Myers and Montgomery, 1995). The statistical approach of Box-Behnken design (BBD) was implied to optimize the integrated approach of BEER process by varying the input factors namely, pH (A), inoculum dosage (B) and contact time (C). Based on the different combination, response, R1 (zinc removal %) was attained. The 3D surface and contour plots of the BBD model is presented in (Fig. 4aef). The output response of the predicted and experimental values is computed by Analysis of variance (ANOVA) that is tabulated in Table 2. As seen from Fig. 4, the 3D surface (Fig. 4aec) and contour plots (Fig. 4def) are found to represent the optimal values of the input variables that shows significant influence towards the zinc removal (%), either as an independent or interaction variable with each other. This is evident with the proper bell-shaped curve that supported the critical relevance with respect to central points. Moreover, the spread of the red region in the contour plots are found to be in perfectly central, thus indicating the significant interaction of the factors towards the response. The model is further validated by two-way ANOVA, that shows the obtained pvalue as <0.0001. This indicates the interaction between the input factors is extremely significant. In case of squared terms, all the three variables are found to have a positive significance towards zinc removal (%). Similarly, the lack of fit F-value (0.41) implies that the lack of fit is not significant with respect to pure error, thus considering the model as fit. Further, the predicted regression coefficient (R2) value of 0.9890 is found to be in a reasonable agreement with the adjusted R2 of 0.9950, showing a difference less than 0.2. In addition, the “Adeq Precision” is a measure of ratio of signal to noise. A ratio >4 is considered desirable. Thereby, the obtained Adeq Precision ratio of 54.283 infers an adequate signal, thus enabling the model to be used for navigating the design space. Based on these statistics, the quadratic coded equation of the BBD can be given as follows, Y ¼ þ 72.34 - 4.88 A þ 4.42 B þ 8.35 C - 9.15 A*B þ 6.35 A*C 8.29B*C - 26.77 A2 - 10.02 B2 - 10.52 C2

(1)

where, Y is zinc removal (%) and A, B, C are pH, inoculum dosage and contact time respectively. The coded equation of the BBD aids in identifying the relative impact/response of the input factors by comparing the factor coefficients. Therefore, the obtained results are found to lie in accordance with statistical values, thereby validating the model for the integrated approach of BEER process for zinc removal studies. A similar study on RSM using central

3.5. Bioleaching vs. BEER approach

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Fig. 4. 3D surface and contour plots showing the interactions between various process parameters, namely, pH, inoculum dosage and contact time with respect to zinc removal percentage.

diminished due to sorption of contaminants onto the soil particles and involvement of H2 and OH ion, generated at the electrodes (Virkutyte et al., 2002). These can be overcome by synergising the bioleaching with electrokinetic process, which was reported to be one of successful and widely applied method of remediation by various researchers (Kim et al., 2010; Lohner et al., 2011). Therefore, we have attempted of imply an integrated approach of bioleaching enhanced electrokinetic remediation (BEER) towards zinc removal from soil was carried out with the optimised process parameters along with an fixed electrical gradient of 20 V direct current (DC) supply and the obtained results are presented in Fig. 5b. From the figure, it can be seen that the process is found to show an increased zinc reduction of 93.08% in much less time than compared to plain

bioleaching and electrokinetic approaches. During BEER process, as the setup was electrified, the zinc content mobilised from the packed soil region at the centre, moved towards the cathode due to concentration gradient near the electrodes. The sulphate part of the ZnSO4 is believed to get released as hydrogen sulphide gas (rotten egg smell), thus leaving the zinc alone in the cathode region. In addition, the optimised process parameters were also found to play a significant role in Zn reduction from the soil, which reflected in form of maximum reduction of Zn than compared to individual processes. The electrolyte in the anodic compartment was maintained at an optimal pH of 5.0 to support the growth of acidophilic bacterium during electromigration. Similar maintenance of the optimised pH in the soil is also believed to favour the effective and

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Table 2 ANOVA for Response Surface Methodology (Quadratic Model)-R1:Zinc removal %. Source

Sum of Squares

df

Mean Square

p-value

Model A-pH B-Inoculum dosage C-Contact time AB AC BC A2 B2 C2 Residual Lack of Fit Pure Error Corr. Total Std. Dev. Mean C.V. % R2 Adjusted R2 Predicted R2 Adeq Precision

5913.42 190.13 156.47 557.45 334.89 161.29 275.23 3018.19 422.61 465.85 13.06 3.09 9.97 5926.48 1.37 50.08 2.73 0.9978 0.9950 0.9890 54.2825

9 1 1 1 1 1 1 1 1 1 7 3 4 16

657.05 190.13 156.47 557.45 334.89 161.29 275.23 3018.19 422.61 465.85 1.87 1.03 2.49

<0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001

significant

0.7532

not significant

Fig. 5. (a) A customized electrokinetic cell set up.1-Titanium electrodes; 2-Filter paper towel; 3-ER area containing Soil þ ZnSO4 þ Serratia marcescens, SMAR1; 4- Aerator plot; and (b) comparative graph showing the percentage of zinc reduction by Bioleaching and BEER approach.

quick removal of Zn (Mulligan et al., 2001; Lavalle et al., 2005; Suzuki et al., 2014). As reported earlier, the lower pH values will enable metal ions such as Cu and Zn become soluble as ionic species in soil (Maini et al., 2000). Overall, the obtained results can be attributed to the synergistic effects of both the bioleaching and electrokinetic process as reported by various other researchers too (Guo et al., 2014; Yang et al., 2016). According to these reports, the integrated process will convert the metal to soluble form (during bioleaching), which favours quick and effective electrokinetic remediation. In an earlier study, a significant reduction of 296.4 mg/ kg to 63.4 mg/kg of Cu and 3756 mg/kg to 33.3 mg/kg of Zn in sewage sludge, within 10 days was reported using indigenous ironoxidising bacteria and EK remediation (Peng et al., 2011). Similarly, in two different studies, Lysinibacillus fusiformis and Pseudomonas putida were employed as a biological component in electrobioremediation technique, by which, an approximate amount of 78% reduction of Hg within 7 days and 89% Zn removal in 5 days

were also reported (Azhar et al., 2016a, 2016b). Thus, the outcome of the present study clearly evidenced the advantage of the integrated process that can overcome these issues of employing individual process of bioleaching and EK remediation. As the spiked soil was used for the present study, microscopic evaluation of the plain soil, spiked soil before and after BEER process was done and the images are presented in Fig. 6a-c. As seen from the figures, the unspiked soil shows clumpy soil aggregates (Fig. 6a), whereas the spiked soil shows an even distribution of zinc throughout the soil sample (Fig. 6b), thus mimicking the real environment in which the zinc is reported to be found in organically bound, exchangeable and water soluble form (Kabeta-Pendias, 1992). In case of Fig. 6c, a noticeable change of disappearance or reduced zinc content in the spiked soil after the BEER process is noted. Thus, these evidences too supported our study of effective and quick reduction of Zn from soil. Therefore, the implementation of an integrated approach of BEER technology employing our

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Fig. 6. SEM micrographs of (a) Soil, (b) ZnSO4 spiked soil before BEER process (c) ZnSO4 spiked soil after BEER process.

acidophilic isolate, Serratia marcescens SMAR1 towards removal of zinc in spiked soil has proven successful in remediating Zn contaminated environment. 4. Conclusions An indigenous acidophilic bacterium was isolated from tannery sludge and molecularly characterized as Serratia marcescens SMAR1 bearing an accession no. MG742410 in NCBI database. A detailed statistical analysis was done to study the interaction between the input factors and response using Box-Behnken design of RSM, Design Expert computer software, v11. Based on the optimised results, comparative studies of bioleaching, electrokinetic (EK) and an integrated approach of bioleaching enhanced electrokinetic remediation (BEER) was carried out using our acidophilic isolate, Serratia marcescens SMAR1. Our isolate was found to exhibit astonishing ability towards Zn reduction in bioleaching process. On the other hand, a maximum Zn reduction of 93.08% was noted in case of BEER process, thus supporting the concept of successful and efficient synergistic effort of bioleaching and EK process in cleaning up Zn contaminated environments. Owing to the results obtained, this technology can be considered as a remediation option to the conventional treatments involving chemical processes. 5. Declarations of interest None. Acknowledgements The authors acknowledges Science and Engineering Research Board, Department of Science and Technology, Government of India for funding this work under N-PDF scheme (File no. PDF/2016/ 002558). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.chemosphere.2018.05.144. References Amiri, F., Yaghmaei, S., Mousavi, S.M., 2011. Bioleaching of tungsten-rich spent hydrocracking catalyst using Penicillium simplicissimum. Bioresour. Technol. 102,

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