Accepted Manuscript Title: Metabolic phasing of anoxic-PDBR for high rate treatment of azo dye wastewater Authors: C. Nagendranatha Reddy, A. Naresh Kumar, S. Venkata Mohan PII: DOI: Reference:

S0304-3894(17)30657-X http://dx.doi.org/10.1016/j.jhazmat.2017.08.065 HAZMAT 18830

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

5-4-2017 18-7-2017 23-8-2017

Please cite this article as: C.Nagendranatha Reddy, A.Naresh Kumar, S.Venkata Mohan, Metabolic phasing of anoxic-PDBR for high rate treatment of azo dye wastewater, Journal of Hazardous Materialshttp://dx.doi.org/10.1016/j.jhazmat.2017.08.065 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Metabolic phasing of anoxic-PDBR for high rate treatment of azo dye wastewater C. Nagendranatha Reddy 1,2, A. Naresh Kumar 1,2, S. Venkata Mohan1,2* Academy of Scientific and Innovative Research (AcSIR)1 Bioengineering and Environmental Science Lab, EEFF Department2, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad-500 007, India * Corresponding author: E-mail: [email protected]; Tel/Fax: 0091-40-27191765 Graphical Abstract BMP Color: 49.5% COD: 44.2 % Acid Black 10B

Aromatic Amines

+

AMP-I Color: 54.4% COD: 56.2 %

O2 Azo reductase

AMP-II Color: 59.4% COD: 62.4 %

+

CO2+ H2O+ NH3

Aromatic Amines

PDBR Biocatalyst

Dye molecule

AMP-III Color: 65 % COD: 69.4 %

Anoxic

Aerobic

Glucose molecule

Highlights    

Strategic induction of air aided the complete mineralization of azo dye Facultative bacteria showed robust activity with varying microenvironments Enhancement in overall performance of anoxic PDBR was achieved in AMPIII strategy Higher azo reductase and dehydrogenase activity correlate well with dye removal

Abstract The treatment of azo dye wastewater was studied in a periodic discontinuous batch reactor (PDBR) at higher loading condition (1250 mg/l) under anoxic microenvironment. The biosystem performance was assessed under different aerobic/total time ratio during the cycle operation [before multiphasing (BMP; Control), 0.014; after multiphasing (AMP): AMPI, 0.84; AMPII, 0.73; AMPIII, 0.65]. Induction of air in anoxically operated PDBR facilitated the simultaneous oxidation and reduction microenvironments and thus resulted in higher dye removal in AMPIII operation (65%) followed by AMPII (59.4%) and AMPI (54.4%) than the

corresponding control operation (BMP: 49.4%). Relatively higher azo reductase enzyme activity was documented with AMP operation thus correlating well with azo dye decolorization. UV-Visible spectra (200-800 nm) resulted significant transformational changes of azo dye peaks (618 nm) before and after multiphase operations. Cyclic voltammograms profiles depicted significant drop in redox catalytic currents during AMPIII operation and also supports the consumption of reducing equivalents towards the dye removal. Derivatives of voltammograms depicted the involvement of various redox mediators viz., cytochrome-C, quinones, Fumarate/Succinate, Fe(CN)63-/Fe(CN)64-, and flavoproteins. Flexibility in phasing the multiple microenvironments in single bioreactor provides new insights in embodying the required capabilities to treat the recalcitrant azo dye wastewater especially at higher dye load operations

Keywords: Periodic discontinuous batch reactor; Redox mediators; Azo Reductase; Dehydrogenase; Wastewater

Nomenclature AMP BMP COD CV DCV DH DSW EA EET FAD Fe(CN)64Fe-S H2S HRT MP MW NAD NADPH OC OLR

After Multiphase Before Multiphase chemical oxygen demand cyclic voltammetry derivative cyclic voltammetry dehydrogenase designed synthetic wastewater electron acceptor extracellular electron transfer Flavin Adenine dinucleotide Hexacyanoferrate Iron Sulphur Hydrogen sulphide hydraulic retention time Multi-phase Molecular weight Nicotinamide adenine Nicotinamide adenine dinucleotide phosphate oxidation current organic loading rate

PBS PDBR RC Rpm Rp SBR SDR SDW SW TTC UV-Vis VFA βa βc

Phosphate buffer solution periodic discontinuous batch reactor reduction current rotations per minute polarization resistance sequencing batch reactor substrate degradation rate synthetic dye wastewater Synthetic waste 2, 3, 5-triphenyltetrazolium chloride Ultra Violet Visible volatile fatty acids oxidative slope Reductive slope

1. Introduction Biological decolorization via anaerobic reductive processes entails co-substrate degradation for liberation of electrons to create the necessary conditions required for -N=N- breakage into corresponding colourless aromatic amines [1-5]. The aromatic amines require aerobic microenvironment for their mineralization through hydroxylation and ring-fission reactions involving non-specific enzymes [2,6]. Hence, sequential integration of micro-aerophilic followed by aerobic processes facilitates decolorization of the dye molecule and complete mineralization of respectively formed aromatic amines. Microenvironment/redox condition plays a significant role in altering the bacterial metabolism and has specific function towards particular pollutant removal and remaining carbon utilization [2,5,7-11]. Tailored metabolic functions facilitates complete mineralization of dye and its intermediates [4,10,12]. Alternative biological process like periodic discontinuous batch (PDBR) mode operation were reported to effectively mineralize dye-based compounds [2,4]. This process is feasible of combining both the required anaerobic and aerobic microenvironments facilitating amalgamation of reductive and oxidative steps in a single reactor [7,11]. Hence, this PDBR has advantages from both biological and engineering point of view aiding in complete biodegradation of azo dyes even at higher dye load operations with no inhibition observed [2,6,13]. Bioelectrochemical treatment [14], membrane reactors [15], bio-electro fenton processes utilizing hydrogen peroxide [16,17], etc. were also employed to treat dye based wastewaters with good degree of successes. Though these processes are efficient, they still have economic and design limitations which need to be resolved for up scaling.

Two or more microenvironments in a single biosystem are requisite to mineralize dye and its intermediates effectively. Induction of anoxic microenvironment in aerobically operated PDBR system helped to facilitate the oxidation and reduction conditions for complete dye mineralization than corresponding single microenvironment operation [5]. Moreover, phase variation PDBR also helps to develop new insights in exemplifying the capabilities of treatment efficiency to address the complex wastewater remediation constrains. In this context, an attempt was made in this communication to evaluate the effect of induced aerobic microenvironment in anoxically operated PDBR by varying the duration of anoxic/aerobic microenvironments in a cycle operation. System performance was critically evaluated for complete mineralization of azo dye by monitoring various process parameters. 2. Materials and Methods 2.1. Dye wastewater An azo dye, C.I. Acid Black 10B [C22H14N6O9S2Na2; 4-amino-5-hydroxy-3-[(4-nitrophenyl) azo]-6-(phenyl azo)-2,7-naphthalene disulfonic acid disodium salt; MW, 616.49; CAS No. 1064-48-8], belonging to acid application class was used as synthetic dye for experiments. Dye (requisite amount; 1250 mg) is dissolved in 1 litre of synthetic wastewater (SW) to prepare simulated dye wastewater (SDW)[4]. Before feeding SDW to the PDBR, pH was adjusted to 7.0 ± 0.2 using 1 N HCl/1 N NaOH. 2.2. Bioreactor Periodic discontinuous batch reactor (PDBR) was designed and fabricated using ‘Perspex’ as material to operate in suspended configuration (total/working volume; 1.0/0.9 l). Peristaltic pump was used to provide uniform mixing of feed with microorganisms. Intermittent sparging (5 min for every 6 h) is provided to maintain micro-aerophilic conditions which also aids in recirculating the feed. 2.3 Biocatalyst Aerobic sludge obtained from secondary settling tank of activated sludge process from the wastewater treatment plant (HMT-Nacharam, Hyderabad) treating domestic sewage was used to inoculate the anoxically operated PDBR. The consortia obtained was washed (phosphate buffer saline; PBS) and re-suspended overnight in designed synthetic wastewater (DSW; 3 g/l glucose). The overnight incubated consortia was transferred (15%) through feed (VSS, 4660 mg/l) into the PDBR. 2.4 Experimental Methodology Initially, the reactor was operated with DSW (without dye; organic loading rate (OLR)- 1.35 kg COD/m3-day) at a hydraulic retention time (HRT) of 48 h in suspended mode. After achieving stabilized and constant performance (COD removal), the reactor was fed with SDW. Previously, the reactor was operated from varying dye load operations (0-1250 mg dye/l), but in the present communication the bioprocess performance was evaluated with 1250 mg dye/l as constant load with the function of tailored microenvironments (Multiphase Approach). Multiphase variations were evaluated during the operation by facilitating consecutive anoxic and aerobic microenvironments in a sequence (Fig 1). The reactor was operated with different phases of operation viz., FILL (15 min), REACT (2820 min), SETTLE (30 min) and DECANT (15 min). All the phases of operation except REACT are constant while anoxic/aerobic microenvironments were varied during the react phase of cycle operation with respect to strategies employed. During BMP (Control) strategy, air was sparged for 5 min (for every 6 h) of react phase while the AMP strategies followed different

time intervals of air sparging as depicted in Figure 1.The aerobic phasing time in the react phase of cycle operation has been varied between 40 min (0.014%; BMP), 2415 min (0.84%; AMPI), 2115 min (0.73%; AMPII) and 1875 min (0.65%; AMPIII) with respect to total time (2880 min). Fig. 1 2.5. Analytical methods Samples collected at regular time intervals were analyzed for various parameters viz., Color, COD, pH, VFA, UV-Vis Spectra (200-800 nm), Enzymes (azoreductase and dehydrogenase), toxicity and cyclic voltammetry analysis. The sample, after centrifugation (2000 rpm; 5 min; 28 oC), was diluted accordingly (50 or 100X) and scanned over 200-800 nm wavelength range using a UV-Vis spectrophotometer (Thermo-Electron) to determine optimum wavelength (λmax 618 nm) for quantitative analysis [4].Other parameters like COD, pH and volatile fatty acids (VFA) were assessed as per the standard methods [18].All the analysis were performed in triplicates and the mean values were represented and discussed. 2.5.1. Toxicity Toxicity analysis was assessed by using Tox-Trak Toxicity test kit (Method 10017; HACH) for both inlet and outlet (treated effluent) samples of each strategy from the PDBR. This procedure is based on reduction of a redox active dye, resazurin, by bacterial respiration and is measured colorimetrically [4]. The percentage (%) inhibition was derived based on the absorbance measured at 610 nm. 2.5.2. Enzymes’ activity The azo reductase enzyme (extracellular) activity was measured colorimetrically at 618 nm [19,20]. The collected samples were analyzed according to the procedure followed in the previous publication [12]. One unit of azo reductase can be defined as the amount of enzyme required to decolorize 1 µmol of dye per minute. The dehydrogenase enzyme activity is estimated based on reduction of 2,3,5-triphenyltetrazolium chloride (TTC), a redox sensitive dye, to non-soluble formazan as a result of respiratory activity. The final resultant mixture obtained was vortexed (200 rpm; 30 min), centrifuged (4000 rpm; 5 min; 28 ± 2 oC) and resulting supernatant's absorbance was measured at 492 nm. 2.5.3. Bio-electrochemical analysis The behavior of consortia under diverse microenvironments during respective reactor operations was evaluated through Cyclic Voltammetry (CV) using potentiostat-galvanostat system (Autolab- PGSTAT12, Ecochemie). Voltamograms recorded at a potential ramp (Range; +0.5V to -0.5 V, scan rate; 30 mV/s) were used for deriving DCV, mediators, tafel plots and slopes and polarization resistance [21,22]. All the electrochemical assays were performed considering a platinum wire as the working electrode and a carbon rod as counter electrode against the reference electrode (Ag-AgCl(S)) using wastewater as electrolyte [5,9,21]. 3. Results and Discussion 3.1 Color Removal Induction of air during REACT phase of Anoxic-PDBR depicted marked influence on the overall performance at higher dye load (1250 mg dye/l). Varying metabolic shifts (AMPI, AMPII and AMPIII) enhanced dye decolourization efficiency compared to control (before multiphase, BMP). In AMPIII operation, the micro-aerophilic and aerobic conditions

dominated in the initial 24 hrs of cycle operation followed by aerobic conditions in the latter stages facilitating sequential integration of reductive (-N=N- breakage into aromatic amines) and oxidative (mineralization of aromatic amines) functions leading to complete mineralization of toxic azo dyes and thereby abetting in higher dye and substrate removal. Prior to multi-phase operation (BMP), Anoxic-PDBR showed color removal efficiency of 49.5%. The aromatic amines formed during the anoxic microenvironment would have inhibited the process efficiency. When the system was directed towards induction of aerobic phases, AMPI operation depicted improvement in color removal efficiency (54.4%) indicating the positive influence of aerobic phases on dye removal (Fig. 2). With the positive influence obtained by inducing short aerobic phases, the duration of air sparging has been increased (AMPII, 59.44%; AMPIII, 65%). The color removed in AMPIII was 31% higher compared to control (BMP) operation depicting the direct correlation of integrative aerobic condition with anoxic operation for complete removal of dye and its intermediates. In the case of cycle operation, maximum color removal was observed within 6 h of cycle operation irrespective of the strategy followed which improved gradually and showed maximum at the end of cycle period. Multiphase operations in aerobically operated PDBR showed good color and COD removal [5]. But in the present study, the performance with respect to color and COD was enhanced by over 7 and 8% respectively depicting the robust activity of facultative inoculum present in the anoxic-PDBR. Fig. 2 The multi-wave scan (200-800 nm) performed at respective time intervals illustrated significant variations in the dye degradation with respect to operation time and strategy followed (Fig 3). Multiwave scan profile provides an idea to analyze the breakdown of N=N- bond present in the azo dye molecule. The spectral analysis revealed a continuous decrement in the intensity of the original dye absorption peak (618 nm) correlating well with the decolorization efficiency observed in initial hours of operation. Untreated azo dye wastewater (Inlet) showed an elevated and defined peaks at 618 and 325 nm corresponding to -N=N- bond and presence of corresponding phenyl and naphthyl rings [5,23,24]. In BMP operation, the peak at 618 nm decreased with time but whereas, the peak at 325 nm has not showed any decrement probably due to not complete breakdown of dye molecule to its intermediates. But in the case of AMP operations, the napthyl ring showed decrement indicating the requirement of aerobic conditions for degradation of complex structures in azo dyes. The absence of peak near UV region illustrates the part or complete cleavage of napthyl rings to form phenyl or aliphatic hydrocarbon intermediates [23]. However, the spectrum range of 320-360 nm corresponds to the presence of aromatic amines resulted from dye degradation [3,22]. Depending on the type of microenvironment, stage of mineralization process and opening of aromatic nuclei, the final products formed may vary from complex aromatic amines, alcohols, aliphatic hydrocarbons to simple NH3, N2, H2O, etc. [25]. The peak at 618 nm showed very less absorbance and almost disappeared in the AMPIII operation which might be due to cleavage of azo bond into respective smaller fragments leading to lower absorbance and formation of slight colored solution [26,27]. Fig. 3 3.2 COD Removal Co-substrate metabolism provides energy for the growth and survival of the microorganisms and generates reducing equivalents which aid in -N=N- breakage (dye degradation) [3,4,12,28]. Electron transfer from co-substrate (primary electron donor) to a final electron acceptor (EA; azo dye) is a rate limiting step in dye degradation through anaerobic process.

Utilization of organics matter for dye removal was represented in terms of substrate degradation rate (SDR, kg COD/m3-day) and COD removal efficiency (%) (Fig. 4). The bioreactor was fed with glucose as co-substrate (organic loading rate (OLR), 1.36 kg COD/m3-day). BMP operation depicted COD removal efficiency of 44.2% (SDR, 0.6 kg COD/m3-day) portraying the inhibition caused by partial removal of toxic dye compounds. Induction of aerobic conditions in the AMPI strategy showed increment in the overall substrate removal efficiency by 12% accounting to 56.2% indicating the positive response of inducing air towards higher COD and color removal. The AMPII strategy documented COD removal of 62.4%. The AMPIII operation documented higher COD removal efficiency of 69.4% which is 25% higher when compared to BMP operation. In line with the dye and COD removal, induction of air depicted affirmative influence on overall SDR. AMPIII operation documented maximum SDR of 0.94 kg COD/m3-day followed by AMPII (0.85 kg COD/m3-day), AMPI (0.76 kg COD/m3-day) and BMP (0.6 kg COD/m3-day). At AMPIII operation, bioreactor documented SDR which was 1.57, 1.24 and 1.11 folds higher than the corresponding BMP, AMPI and AMPII operations respectively. In BMP and AMPI operations, the COD removal was observed maximum till 24 h of cycle operations but whereas AMPII and AMPIII operations showed COD removal till the end of cycle operation depicting the necessity of inducing aerobic conditions throughout the cycle for COD and/or organic compounds removal. Fig. 4 3.3 Toxicity The dye molecule with the help of biocatalyst is cleaved to respective aromatic amines which are further more toxic to environment rather than the intact dye molecule. Hence, the toxicity analysis was performed to evaluate the amount of toxicity removed in the bioreactor in all the strategies followed to enhance the treatment efficiency. The % inhibition is determined by comparing the response given by a control solution to that of corresponding dye sample [5]. Prior to feeding, inlet (1250 mg dye/l) depicted 97% of inhibition which is because of higher dye concentration illustrating the % inhibition is directly proportional. Higher removal in % inhibition was observed in AMPIII (40.8%) operation followed by AMPII (51.4%), AMPI (60.2%) and BMP (70.4%) operations (Fig. 5). In AMPIII operation, the % inhibition reduction is 2.38 and 1.73 folds when compared to inlet and BMP operations respectively. Significant reduction in % inhibition was observed after multiphasing. Both color and COD removal in AMP strategies supports the toxicity reduction in the final outlet from the bioreactor. Fig. 5 3.4 Enzyme Activity 3.4.1 Azo reductase Azo dye biodegradative pathways were initiated by azo reductase enzyme, produced in situ by enriched microbiomes, catalyzes the reductive cleavage of azo bond (-N=N-) using different reduced metabolites or redox mediators [19,29]. The enzyme activity was observed to increase with strategic anoxic/aerobic phasing during initial and latter stages of cycle operation which showed direct correlation with dye decolorization. BMP documented enzyme activity of 26.1±0.18 U which was lower than the corresponding multiphase operations (AMPI: 30.9±0.09 U, AMPII: 32.8±0.12 U; AMPIII: 36.5±0.3 U) (Fig. 6). Azo reductase activity by 1.4 folds was observed in AMPIII operation compared to conventional BMP operation. The protons and electrons liberated by the conversion of NADH to NAD are

utilized by the dye for its reduction to hydrazines and aromatic amines respectively [14]. With the adaptation of biocatalyst to dye environment, the enzyme activity attained higher values during the initial phase of the cycle operation (within 6 h) in all the strategies depicting the robustness of biocatalyst for the complex carbon intake. In BMP operation, at 6 h time interval, enzyme activity of 16.6 U was observed for 29.72% of dye decolorization which constitutes about 60% of overall dye removal. But whereas in AMP operations, the enzymes activity of 14.5 U (4 h), 15.1 U (4 h) and 18.4 U (6 h) was observed for 46% (AMPI), 45 % (AMPII) and 47% (AMPIII) dye decolorization efficiency respectively depicts the significant role of enzyme in the cleavage of azo compounds and correlates well. With the course of operation, drastic improvement in the enzyme activity was observed at the initial phase (till 24 h) followed by gradual improvement at the end of the cycle operation (24-48 h). Fig. 6 3.4.2 Dehydrogenase The dehydrogenase enzyme, belonging to oxido-reductase group, catalyzes the redox reactions for metabolite inter-conversion intracellular and serves as a microbiological redox indicator representing metabolic activities of microorganisms [30]. Distinct variations in the DH activity were observed with the function of varying microenvironments during cycle operation. Higher DH activity was observed with AMPIII operation (3.28 µg/ml; 25 h), followed by AMPII (3.11 µg/ml; 24 h), AMPI (2.89 µg/ml; 24 h) and BMP (2.51 µg/ ml; 24 h) indicating the enhanced metabolic activity during strategic aeration provided to the PDBR (Fig. 7). Under AMPIII operation, almost stable and higher DH activity was observed throughout the study. This might be attributed to the availability of more H+ from substrate oxidation. With the help of redox mediators’ viz., NAD+, FAD+, DH facilitates transfer of protons from organic substrates to inorganic acceptor during metabolism. The enzyme activity was observed to increase with increase in the intermittent aerobic phase. In contrast to azo reductase activity, the DH activity showed increment till 24 h of cycle operation followed by decrement thereafter by the end of the cycle as evidenced in previous studies [4,5]. Higher dye and substrate removal observed with AMPIII operation also correlated well with the DH activity during the cycle reaction time. DH plays a significant role in dye reduction to its intermediates by catalyzing the proton transfer [21]. Fig. 7 3.5 Redox Microenvironment Analysis 3.5.1 Volatile fatty acids VFA are important metabolic by-products that get released through substrate reduction [31]. VFA synthesis co-exists with the anaerobic metabolic pathway due to the process of reductive acidogenesis. In BMP operation, the amount of VFA concentration increased with respect to time and reached maximum (1152 mg/l; pH 5.11) by the end of cycle operation. The increment in VFA during the later phase of operation indicates the higher and controlled substrate degradation. But in AMP operation, the VFA concentration varied in an undulating pattern with respect to microenvironment. AMPI operation showed higher VFA production compared to other AMP operations (24 h; 1084 mg/l; pH 5.5). VFA concentration was higher in case of AMPIII operation (1108 mg/l; 14 h; pH 5.55) during initial cycle depicting anoxic conditions and showed decrement (121 mg/l; 43 h; pH 7.87) during latter stages depicting the prevalence of higher aerobic conditions. Concomitant increase and decrease in VFA concentration during the operation might be attributed to the induced anoxic and aerobic microenvironments persisting during the initial and final phase of cycle operation. VFA in the presence of protons and dye intermediates function as redox mediator and shuttles electrons

from carbon (electron donor) to azo dye (terminal electron acceptor) [32]. The hydrogen generated along with VFA (formate) may function as possible electron transfer carrier for syntrophic degradation [33]. Fig. 8 3.5.2 pH pH profiles depicts the exact mirror images of corresponding VFA profiles. As the metabolic shifts were varied consecutively with respect to time interval, the facultative conditions prevail in the bioreactor influencing the overall system activity. The BMP operation crafts the biocatalyst to follow anaerobic environment thereby resulting a drop in system pH to 5.11 by the end of cycle operation (Fig. 8). But whereas in AMP operations, pH was observed to follow an undulating pattern representing the consecutive metabolic shift variation followed in three different strategies. A maximum acidification was observed with AMPI and AMPIII (pH ~5.5). The bioreactor evidenced lowest pH of 5.11 (48 h) and highest pH of 7.87(43 h) during BMP and AMP III operations respectively. The drop in system pH might be due to the higher substrate activation leading the generation of more acid intermediates. AMP operations depicted pH shift towards basic condition during the end of cycle operation due to the prevailing aerobic conditions. AMPIII operation depicted pH drop in the initial stages followed by increment in pH during latter stages of cycle operation. 3.6 Bioelectrochemical Analysis Voltammograms visualized a marked variation in redox catalytic currents (RC: reduction currents, OC: oxidation currents) with the function of bioreactor operation (Fig. 9). Relatively, higher redox catalytic currents were observed with AMPIII operation (OC/RC: 0.26/-1.13 A) in comparison to AMPII (0.33/-0.29 A), AMPI (028/-0.28 A) and BMP (0.19/-0.14 A) operations. AMP operations showed gradual increment in redox catalytic currents after inducing strategic aeration to the PDBR indicating the direct positive effect that might have entrusted the biocatalyst capability towards dye reduction and substrate oxidation. The OC of BMP, AMPI and AMPII operations showed distinct variations with respective time interval with no significant changes in RC. But in case of AMPIII operation, the RC showed significant variations compared to OC which is in contrast to other strategies. The dye at higher concentration functions as oxidizing agent (electron acceptor) and has more tendency to accept electrons and get reduced into its corresponding amines. Fig. 9 3.6.1 Bioelectrokinetics Tafel plots also helps in understanding the shift in biocatalyst's redox behaviour in the bioreactor. In BMP operation, significant shift towards more negative potential (-0.4 V) was observed but whereas in AMP operations, shift was observed towards oxidation/midpoint (towards 0 V) by initial hours (6 & 12 h) of cycle operation favouring higher reduction in the system. The lower negative potential is considered as an effective sign of low free energy requirement for occurrence of reduction reactions making the process (dye degradation) viable by complete mineralization of dye molecule. The higher redox reactions occurring in the AMP operations illustrate the feasibility of varying microenvironments for dye reduction and oxidation of intermediates by accepting the liberated electrons. Tafel analysis was employed to study the bio-electro kineticbehaviour of biocatalyst based on the oxidative Tafel slope (βa), reductive Tafel slope (βc) and polarization resistance (Rp). Oxidation and reduction reactions help to understand the biocatalytic activity and losses occurring in the system. BMP operation showed higher βa (0.24 V/dec) and βc (0.721 V/dec)

compared to AMP operations (βa: 0.098 V/dec; βc: 0.32 V/dec) indicating the favourability of high redox reactions in the AMP liberating more number of electrons towards dye reduction. The lower slopes with AMP operation supports the functionality of anoxic microenvironment in delivering the electrons at a higher rate with the requirement of less energy. AMPIII operation showed rapid decrement till 36 h (0.098 V/dec) indicating the occurrence of oxidation reaction till 36 h which is in correlation with substrate degradation (Fig. 10). In addition, lower reduction slope observed at both the microenvironments supports the effective utilization of electrons towards dye reduction as well as the function of the electron carriers in neutralizing the dye intermediates. This in turn supports the less requirement of activation energy leading to less electron losses in the multiphase microenvironment towards dye removal. Polarization resistance (Rp) derived from Tafel analysis helps to understand the resistance observed during the electron transfer to dye molecules [21]. Rp was found to be less in AMP than BMP signifying the impact of the employed multiphase strategy towards dye degradation presuming less resistance in electron delivery. BMP operation depicted higher Rp of 1.26 Ω (0 h) followed by AMPII (1.07 Ω; 0 h), AMPI (1.04 Ω; 24 h) and AMPIII (0.86 Ω; 12 h). This low Rp of AMP operations signifies the effective transfer of electron for dye degradation with minimal losses which is in contrast to corresponding BMP where there is possibility of insufficient oxygen for dye intermediate oxidation in the bioreactor. Fig. 10 3.6.2 Redox Mediators Signals for the redox shuttles (mediators) will appear on the voltammetric signature, when the redox potential of a mediator equals to the applied potential. Mediators are able to quickly diffuse in and out of enzymatic channels thus shuttling electrons from enzyme active site to terminal electron acceptor (dye) thereby enhancing the dye degradation. Different peaks were observed on the voltammograms under anoxic and aerobic microenvironments during BMP and AMP operations (Table 1). Cytochromes were detected during all the operations which plays a major role in extracellular electron transport chain (EET) and carries an electron to TEA (dye) which is also capable of undergoing redox reactions [34]. Fumirate/Succinate, Quinones and Fe(CN)63-/Fe(CN)64- are detected in few other experimental variations apart from cytochromes. Table 1 3.6.3 Derivative analysis The derivative cyclic voltammetry represents the rate of change of the voltammetric current with respect to time and electrode potential E(di/dt). This DCV provides higher number of mediator peak potentials when compared to normal CV analysis [22]. Distinct peaks were observed in AMP operations at each time interval during the cycle operation when compared to BMP (Table 2). In BMP operation, peaks corresponding to Flavoproteins, quinones, Cytochromes, Fumarate/Succinate, Fe-S proteins and SO42-/H2S or FAD+/FADH2 were detected. Fe-S proteins have the capability to transfer only one electron from the biocatalyst to the dye molecule but the flavoproteins functions as a specific electron acceptor for primary dehydrogenases, transferring the electrons to terminal respiratory systems such as electrontransferring-flavoprotein dehydrogenase [35]. AMP operations detected mediators mostly belonging to Cytochrome complex but others like quinones, Fumarate/Succinate, Fe(CN)63/Fe(CN)64-, Flavoproteins and Fe-S proteins were also detected during the operation (Fig. 11). The presence of NO3-/NO2- during the operation depicts the presence of end products formed during the complete mineralization of dye molecule and acting as electron shuttles [26]. All

the redox mediators detected during DCV analysis are the membrane bound proteins of bacteria that act as electron carriers during the process (Table 2). Fig. 11 & Table 2 Finally, PDBR operation has the advantage of integrating two requisite microenvironments in single-system operation for simultaneous function of oxidation and reduction. Mixed facultative consortia present in the PDBR effectively decolorized the recalcitrant dye at elevated dye loads. Contribution of aerobic conditions on overall system performance is significant which is evidenced by overall PDBR performance. This multiphasing provides new insight in embodying the capabilities of the SBR operation and degradation of toxic and higher concentrations of pollutants. 4. Conclusions  The complete degradation of Acid Black 10B was clearly demonstrated by sequential integration of anoxic and aerobic microenvironments  Biological degradation of higher dye load (1250 mg/l) in a single PDBR system is first of its kind  Inducing aerobic condition facilitated system microenvironment to switch between oxidation and reduction assisting the reduction of dye followed by the mineralization of intermediates.  Experimental results suggests that majority of the color removal (-N=N- cleavage) occurred in the anoxic stage but whereas higher COD removal was observed in aerobic conditions  By increasing anoxic conditions initially followed by more aerobic conditions in latter stages of cycle operation showed significant effect on pollutant removal by the same mixed bacterial culture  AMPIII strategy showed higher color/COD removal along with enzymes when compared to BMP operation  The results of UV-Vis spectra and Toxicity demonstrated the reductive cleavage of azo bond under anoxic conditions and mineralization of amines under aerobic conditions facilitating complete mineralization of toxic dye compounds  DCV analysis detected various redox shuttles’ during all the MP strategies depicting higher dye reduction by minimizing the losses.  Lower Tafel slopes documented with AMPIII operation illustrate higher electron transfer reactions with losses minimization Acknowledgements The authors wish to thank the Director, CSIR-IICT for encouragement in carrying out this work. Funding from Council for Scientific and Industrial Research (CSIR - India) in the form of XII five year network projects [SETCA (CSC-0113)] is gratefully acknowledged. CNR and ANK duly acknowledge CSIR, New Delhi, for providing research fellowship.

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Figure Captions Figure 1: Schematic representation of multiphase variation strategies followed to enhance overall performance of PDBR Figure 2: Color removal efficiency of one best cycle (Cycle 6) with respect to strategies employed and time intervals Figure 3: (a) UV-Vis Spectrum (200-800 nm) of various strategies followed (b) Comparison of UV-Vis Spectrum with respect to various time intervals (6, 12 and 48 h) during cycle operation Figure 4: (a) COD removal efficiency of one best cycle with respect to strategies employed and time intervals; (b) Comparison of COD removal concentration and SDR Figure 5: (a) Toxicity profiles (b) overall performance of PDBR reactor with respect to diverse strategies followed Figure 6: Azoreductase activity depicted with respect to various strategies employed Figure 7: Dehydrogenase activity with respect to strategies employed and time intervals Figure 8: pH and VFA profiles depicted with respect to strategy followed and time intervals Figure 9: Cyclic voltammogram (CV) profiles recorded during PDBR operation with respect to strategies employed Figure 10: (a) Tafel plots derived from CV (b) Oxidative, reductive Tafel slopes and polarization profiles. Figure 11: Derivative cyclic voltammogram (DCV) profiles recorded with respect to various strategies employed

DECANT

AMPIII AMPII AMPI BMP

Figure 1

Color Removal Efficiency (%)

60

40

20

0

BMP AMPI AMPII AMPIII 0

6

12

18

24

30

Time (h)

Figure 2

36

42

48

0.7

a

Absorbance (OD)

0.0

BMP 0h 36h

6h 48 h

12h

24h

AMPI

0.7 0.0

AMPII

0.7 0.0

AMPIII

0.7 0.0 200

300

400

500

600

700

800

Wavelength (nm) 1.2

Inlet AMPII

b

BMP AMPI AMPIII

6h

Absorbance (OD)

0.6 0.0 1.2

12 h

0.6 0.0 1.2

48 h

0.6 0.0 200

300

400

500

600

Wavelength (nm)

Figure 3

700

800

COD Removal Efficiency (%)

40

20

0

BMP AMPI AMPII AMPIII 0

6

12

18

24

30

36

42

48

Time (h)

3000

1.2

BMP (COD) AMPI (COD) AMPII (COD) AMPIII (COD)

b

2500

SDR (Kg.COD/m -day)

0.8

2000

3

COD Concentration (mg/l)

a

60

0.4

1500

BMP (SDR) AMPI (SDR) AMPII (SDR) AMPIII (SDR)

1000 0

6

12

18

24

30

Time (h)

Figure 4

36

42

48

0

Toxicity (% Inhibition)

100

a

80

60

40

20

0

Inlet

Removal (%)

80

BMP

AMPI

Color

AMPII

COD

AMPIII

Toxicity

b

60

40

20

0

Figure 5

BMP

AMPI

AMPII

AMPIII

Azoreductase Activity (U)

40 35 30 25 20 15 10

BMP AMPI AMPII AMPIII

5 0

0

6

12

18

24

30

Time (h) Figure 6

36

42

48

BMP AMPI AMPII AMPIII

Dehydrogenase Activity (g/ml of Toluene)

3

2

1

0

6

12

18

24

30

Time (h) Figure 7

36

42

48

AMPI

8 BMP

900 600

6

pH

5 4 8 AMPII

300

pH VFA

AMPIII

900

7

600

6

300

5 4

0

6 12 18 24 30 36 42 48 0

Time (h)

Figure 8

0

6 12 18 24 30 36 42 48

0

VFA Concentration (mg/l)

7

i / i/A

0.03u

0.2u i / A

A

6h 12 h 24 h 48 h 0h 36 h

0.3u 0.1u 48 h

i/A

0.20u

0 0h

36 h

-0.1u 24 h -0.2u 12 h

6h

BMP

-0.15u -0.75 -0.50 -0.25 0 0.25 0.50 0.75

AMPI

-0.3u -0.750 -0.500 -0.250 0 0.250 0.500 0.750 E / V Vs Ag/AgCl (S)

E/V Vs Ag/ AgCl (S) Figure 9

i / A

i/A

6h 0.3u 12 h 24 h 0.2u 36 h 0h 0.1u 48 h -0.1u -0.2u AMPII -0.3u -0.75 -0.50 -0.25 0 0.25 0.50 0.75

i / i/A

A

E/V Vs Ag/AgCl (S) 0.50u 0.25u 36 h 0 48 h -0.25u 0 h -0.50u 24 h -0.75u 6 h -1.00u 12 h AMPIII -1.25u -0.750 -0.500 -0.250 0 0.250 0.500 0.750 E / V Vs Ag/AgCl (S)

-15

a

-20

ln I

-15 -20

0h 24 h

BMP

6h 36 h

12 h 48 h

AMPI

-15 -20 -15

AMPII

-20 AMPIII

-0.4

-0.2

0.0

0.2

0.4

0.6

E/V

Rp () c (V/dec)

aV/dec)

b

BMP AMPII

0.2

AMPI AMPIII

0.1

0.6

0.3

12.0M 6.0M

0

6

12

18

24

30

Time (h)

Figure 10

36

42

48

100n

0h 24 h

6h 36 h

12 h BMP 48 h

di/dt / A/s

0 60n

AMPI

0 700n

AMPII

0 100n

AMPIII

0

-0.6

-0.4

-0.2

0.0

0.2

E/V Vs Ag/AgCl (S)

Figure 11

0.4

Table 1: Redox Mediators observed during CV Analysis CV Analysis Mediators Time Peak Mediator/Redox Position Shuttle* (h) 6

0.129

Cytochrome bc1

24

0.36

Fe(CN)63-/Fe(CN)64-

6

0.383

Cytochrome aa3

12

0.302

Cytochrome C

24

0.332

Cytochrome C

0

0.175

Cytochrome bc1

6

-0.05

Quinones

12

0.035

Fumirate/Succinate

24

0.25

Cytochrome C ox/red

36

0.117

Cytochrome bc1

0

0.36

Fe(CN)63-/Fe(CN)64-

0.277

Cytochrome C

0.03

Fumirate/Succinate

0.2

Cytochrome bc1

0.344

Cytochrome C

0.305

Cytochrome C

0.033

Fumirate/Succinate

0.2

Cytochrome bc1

0.04

Cytochrome bc1

0.2

Cytochrome bc1

BMP

AMPI

AMPII

6

12 AMPIII 24

36 48

*[17,31]

Table 2: Redox Mediators detected during DCV Analysis DCV Analysis Mediators Time (h)

Peak Position

Mediator/Redox Shuttle

0.262

Flavoproteins

-0.03

Quinones

0.302

Cytochrome C

0.03

Fumirate/Succinate

-0.101

Fe-S Proteins

0.04

Cytochrome bc1

0.312

Cytochrome C

0.272

Cytochrome C

-0.222

SO42-/H2S/FAD+/FADH2

0.262

Flavoproteins

-0.05

Quinones

0.181

Cytochrome bc1

0.171

Cytochrome bc1

-0.05

Quinones

0.282

Cytochrome C

0.312

Cytochrome C

0.171

Cytochrome bc1

0.232

Cytochrome bc1

0.232

Cytochrome bc1

0.252

Cytochrome C ox/red

0.282

Cytochrome C

0.211

Cytochrome bc1

-0.05

Quinones

0

6

12

BMP 24

36

48

0

6 AMPI 12 24

36

0.211

Cytochrome bc1

0.252

Cytochrome C ox/red

-0.05

Quinones

0.181

Cytochrome bc1

0.201

Cytochrome bc1

0.171

Cytochrome bc1

0.181

Cytochrome bc1

0.131

Cytochrome bc1

0.312

Cytochrome C

0.201

Cytochrome bc1

0.282

Cytochrome C

0.282

Cytochrome C

0.035

Fumirate/Succinate

0.211

Cytochrome bc1

0.366

Fe(CN)63-/Fe(CN)64-

0.235

Cytochrome bc1

0.188

Cytochrome bc1

0.2

Cytochrome bc1

0.2

Cytochrome bc1

0.211

Cytochrome bc1

0.117

Cytochrome bc1

0.305

Cytochrome C

0.2

Cytochrome bc1

0.312

Cytochrome C

0.252

Cytochrome C ox/red

0.282

Cytochrome C

48

0

6

12 AMPII

24

36

48

0 AMPIII 6

12

24

0.04

Cytochrome bc1

-0.151

Flavoproteins

-0.141

Fe-S Proteins

0.201

Cytochrome bc1

0.386

Cytochrome aa3

0.141

Cytochrome bc1

0.36

Fe(CN)63-/Fe(CN)64-

0.43

NO3-/NO2-

0.2

Cytochrome bc1

0.211

Cytochrome bc1

0.04

Cytochrome bc1

36 48 *[17,31]

Metabolic phasing of anoxic-PDBR for high rate treatment of azo dye wastewater.

The treatment of azo dye wastewater was studied in a periodic discontinuous batch reactor (PDBR) at high loading condition (1250mg/l) under anoxic mic...
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