Improvement in treatment of soak liquor by combining electro-oxidation and biodegradation

Abstract

Soak liquor generated from the leather processing industry contains high organic load, making it a challenge to treat it efficiently. A combined process involving electro-oxidation and biodegradation by halophilic bacteria was applied to treat wastewater effectively for discharge. Electrolysis was performed prior to biological treatment in an electrochemical reactor at a current density of 0.012 A cm⁻² for a period of 30 minutes. Titanium Substrate Insoluble Anode (TSIA) was used as an anode and titanium mesh was used as a cathode. Biological degradation was carried out with two isolated microbial strains at pH 6. The combination of electro-oxidation and biodegradation was recorded as a good technique in terms of COD (Chemical Oxygen Demand), BOD (Biological Oxygen Demand) and TKN (Total Kjeldahl Nitrogen) removal efficiency with values of 95%, 85%, and 88% respectively. The present study claims that the integrated process gives better performance for the reduction of COD when compared to previous studies.

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1 Introduction

Soak liquor, a primary effluent from the tannery industry has an intense brown colour, hypersalinity, and stinky odour. Soak liquor is composed of 2–4% sodium chloride by weight and contains traces of calcium chloride along with organic contaminants (flesh, skin, blood, humic substances and other suspended particles) such as nitrogen and phosphorous containing compounds.^1,2 If this effluent is discharged into nearby lands without any prior treatment, it will pollute the lands and groundwater. The treatment method in use is solar evaporation but the evaporation rate is affected due to higher total dissolved solids (TDS), organic loads and suspended solids (SS).³ The other additional problem of this effluent treatment is the presence of humic substances. These compounds form complexes with proteins and other organic contaminants making it difficult for biological degradation.⁴ The breaking down of these complexes is necessary for effective biological treatment of soak liquor. Some reports also indicated that bacteria found in the hypersaline environment have greater potential in degrading pollutants.^3,5 The native concentration of chloride favours pre-treatment in electro-oxidation. Szpyrkowicz et al.,⁶ Srinivasan et al.⁷ and Kanagasabi et al.⁸ did pre-treatment processes viz. electro-oxidation and chemical oxidation (ozone treatment) before biological treatment. The electrochemical pre-treatment was used to convert bio-recalcitrant organics to biodegradable compounds.⁹ The secondary oxidants such as chlorine and hypochlorite liberated during the electro-oxidation process enhance the biodegradability, resulting in the removal of organic pollutants in the effluent. Basha et al.² stated that the rate of biological treatment can be enhanced by reducing the molecular size of the organic pollutant present in the effluent by electro-oxidation, where TSIA (titanium substrate insoluble anode) was used as the anode. Sundarapandiyan et al.¹⁰ performed electrochemical oxidation of synthetic saline wastewater by employing graphite electrodes and suggested that 0.012 A cm⁻² is the optimum current density for the treatment of saline wastewater with a COD reduction of 89.11% within 2 h where biological degradation was not investigated.

In recent years, a few studies (Table 1) have been carried out on combined processed viz. electro-oxidation and biological treatment of tannery soak liquor, where the COD reduction was in the range between 64% and 90% [(76%),⁶ (64%),⁷ (66.2%),⁸ (90%),¹¹] where the current density was 0.02 & 0.04 A cm⁻²,⁶ 0.05 A cm⁻²,⁸ 0.024 A cm⁻² (ref. 11) respectively in electro-oxidation. In some of the studies on biological degradation alone in soak liquor, the COD reduction was about 96% in 300 days,³ 74 to 88% in 45 days⁴ and 80% in 72 h.¹² Senthilkumar et al.¹² used halophilic bacteria collected from marine and tannery saline wastewater for degradation of soak liquor and the COD removal efficiency was about 80% within 3 days whereas the initial COD was about 2512 ppm in raw tannery saline wastewater.

Table 1 Previous studies on degradation of tannery effluent

Reference	Type of effluent	Initial COD (ppm)	COD removal efficiency (%)	Single/integrated process	Remarks
2	Soak liquor	5800	94.8%	Electrochemical method	Current density −0.058 A cm^−2; experiment: 7.05 h
3	Soak liquor	1500–4400	96%	Biological method (combined anaerobic and aerobic process)	Duration of the experiment: 300 days
4	Tannery wastewater	4800 ± 350	74–88%	Biological method	Duration of the experiment: 45 days
6	Raw wastewater	2386	76%	Integrated process (electrochemical and anaerobic process)	Current density – 0.02 and 0.04 A cm^−2; electrode selection Ti–Pt–Ir and Ti/PdO–Co3O4
7	Primary tannery effluent	890–1600	64%	Combined (chemical and biological oxidation)	Duration of the experiment: 36 h
8	Chrome tannery	4600–6000	66.2%	Combined (electrochemical and biological oxidation)	Current density – 0.05 A cm^−2; duration of the experiment: EO – 90 min and biodegradation – 7 days
10	Soak liquor	3000–6000	89.11%	Electro-oxidation	Current density – 0.012 A cm^−2; duration of the experiment: 2 h
11	Soak liquor	4466	90%	Combined (electrochemical and photovoltaic stand-alone system)	Current density: 0.024 A cm^−2; duration of the experiment: 3 days
12	Saline wastewater	2512	80% in 8% salinity	Biological method	Duration of the experiment: 48 to 72 h
13	Evaporated residue of soak liquor	5570 ± 0.04	75%	Electrochemical method	Current density: 0.05 A cm^−2; duration of the experiment: 4 h
Present study	Tannery soak liquor	7300 ± 0.10	96%	Combined (electrochemical and biological)	Current density: 0.012 A cm^−2; duration of the experiment: EO – 30 min and biodegradation – 7 days

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In the present communication, the importance of electro-oxidation prior to biological treatment of soak liquor containing high COD was explored. Electrochemically generated secondary oxidants were removed by solar exposure. The combined process viz. electro-oxidation and biological treatment using (electro-biodegradation) halophilic bacteria was investigated to improve the COD reduction. The initial COD of the soak liquor was about 7300 mg L⁻¹ which is the highest when compared to the previous works presented in Table 1. The soak liquor was electro-oxidized with low current density (0.012 A cm⁻²) compared to previous works using triple oxide coated electrode (TSIA) to break the humic acid organic complex followed by biodegradation using Bacillus cereus and Klebsiella oxytoca with constant agitation (150 rpm). The removal of organic contaminants during the process was investigated by monitoring COD, BOD, TKN, lipid, and protein removal efficiency before and after electro-biodegradation.

2 Materials and methods

Soak liquor was collected from the leather processing division, CSIR-Central Leather Research Institute (CLRI), Chennai and transported in an icebox to the CSIR-Central Electrochemical Research Institute (CECRI) Karaikudi. The sample was filtered using Whatman No. 1 filter paper to avoid the contamination of suspended particles such as hair, sand etc., present in the soak liquor. The filtered particles were discarded carefully and the filtered soak liquor was further used in the study.

The physical and chemical properties of filtered soak liquor were characterized using standard methods.¹³ COD and BOD were determined using the dichromate open reflux method and Winkler’s method respectively by strictly following the American Public Health Association (APHA) procedures.¹³ The interference of chloride during COD measurement was overcome by adding 10 L⁻¹ weight ratio of mercuric sulphate to chloride.³ Proteins¹⁴ and lipids¹⁵ were measured before and after electro-biodegradation using standard estimation procedures. Electro-bio degraded samples were centrifuged at 6000 rpm for 30 minutes and the supernatant was used for protein and lipid estimation in order to avoid the interference of cell suspensions.

2.1 Electro-oxidation of soak liquor

2.1.1 Electrodes. IrO₂–RuO₂–TiO₂ film coated titanium expanded mesh prepared by a thermal decomposition method was used as the anode. The respective metal chloride was dissolved in isopropyl alcohol and brushed on the pre-treated titanium substrate layer by layer until the required coating solution was brushed on the titanium.^16,17 After brushing each layer, the substrate was heated in a furnace at 450 °C in the presence of air. The coating consists of TiO₂: 76%; RuO₂: 23%; IrO₂: 1% and the coating was characterized by SEM and EDAX and presented in ESI Fig. 1 and 2 and Table 1.† Titanium expanded mesh was used as the cathode.

A rectangular undivided cell of dimension 15 × 5 cm² was designed and fabricated using polypropylene solid material with a volume of 350 mL with a TSIA anode and titanium mesh cathode. The anode and cathode were kept apart at an interelectrode distance of 1 cm. The soak liquor was taken into the cell and electrolysed at a current density of 0.012 A cm⁻² for 30 minutes. The experiments were done at a galvanostatic condition using a DC power supply (Aplab power supply model: regulated DC power supply L3205). Anodic and cathodic potentials were measured using a multimeter (Agilent U1232A) with the help of saturated calomel electrode (SCE) as a reference electrode. The excess hypochlorite present after electro-oxidation was decomposed by exposing to sunlight for 1 h at 1.0 × 10⁵ lux as measured by Lux meter (Digital 128 instruments). The same electrode was used to treat multiple batches (6 times) of soak liquor to find the fouling and the nature of coatings on the electrode surface.

2.2 Isolation and identification of the bacterial strain

The bacterial strains were isolated from the soak liquor. The isolated bacterial strains were enriched in nutrient agar medium.⁸ Pure cultures were obtained by picking morphologically dissimilar and dominant isolates, which were stored at 4 °C with periodic subculturing. These strains were identified using 16s rRNA sequencing in Defence Research and Development Organisation (DRDO), Gwalior.

2.3 Electro-biodegradation of soak liquor

The isolated bacterial strains were inoculated in hypochlorite free electro-oxidized soak liquor (250 mL) supplemented with 1% of glucose as the energy source.¹⁸ The electro-oxidized soak liquor was inoculated with 5% of a 5 day old culture and agitated at 150 rpm at 30 °C. The growth was monitored by measuring the optical density at 600 nm using a UV-spectrophotometer (Thermo Scientific Evolution 201). Growth conditions such as pH (4 to 9), temperature (25 °C to 40 °C), inoculum concentration (5% to 25%), the percentage of the carbon source (1% to 5%) and agitation (100 rpm to 250) were optimized by measuring the biomass concentration at 600 nm. The condition for the biodegradation study was chosen based on higher yielding biomass.

2.4 Decolourization and degradation studies

UV-Vis spectra were recorded between 190 and 1100 nm using UV-Vis spectrometer (Thermo Scientific Evolution 201) and the spectrum was compared for soak liquor before and after electro-oxidation and biological degradation. The colour removal efficiency and organic load removal pattern were analysed. The variation in the functional group during the electro-oxidation and biodegradation were determined using FT-IR (Bruker Optik GmbH, model no – Tensor 27). The samples were mixed with KBr and the transmittance percentage was observed between a wavenumber range of 400–4000 cm⁻¹. The variations in the percentage of hydrogen, nitrogen and sulphur content of the soak liquor, before and after electro-oxidation and electro-biodegradation were measured by CHNS (Carbon, Hydrogen, Nitrogen, Sulphur) analysis (Elementar Cube Vario). The soak liquor before and after electro-oxidation and biodegradation was lyophilized and used for high-pressure liquid chromatography (HPLC) analysis. 20 μL of the sample was injected into a liquid chromatographic system (Prominence, Shimadzu) with a photodiode array (PDA). The chromatographic separation was performed on a C18 column (100 mm × 4.6 mm) using methanol/water (acidified 1%, v/v) as the mobile phase at a flow rate of 0.4 mL min⁻¹.¹⁹

2.5 Energy consumption

Energy consumption during the electro-oxidation of soak liquor was calculated on the basis of COD reduction:²⁰
where t is the time of electrolysis in hours, V is the average cell voltage, A is the current in ampere, SV is the sample volume in litres and ΔCOD is the difference of COD in time t in mg L⁻¹.

3 Results and discussion

The physiochemical properties of soak liquor during various stages of the process are mentioned in Table 2. The soak liquor contained protein (13 g L⁻¹), lipid (89 g L⁻¹) and chloride (17.08 g L⁻¹) as major components where the COD was 7300 mg L⁻¹.

Table 2 Physiochemical properties of soak liquor^a

Parameters	Soak liquor	E.O.S	E.O-ST	HF-E.O-BT
a E.O.S – electro-oxidized soak liquor, E.O-ST – electro-oxidized soak liquor after solar treatment, HF-E.O-BT – hypochlorite free soak liquor after biological treatment.
pH	7.9 ± 0.6	7.75 ± 0.4	7.61 ± 0.3	3.2 ± 0.4
Colour	Intense brown	Pale yellow	Pale yellow	Transparent
Odour	Foul smell	Bleach smell	Nil	Nil
Protein (g L^−1)	13 ± 0.2	12 ± 0.2	11.64 ± 0.2	5.5 ± 0.2
Lipid (g L^−1)	89 ± 0.2	61 ± 0.2	60.86 ± 0.2	7.6 ± 0.2
TKN (mg L^−1)	420	150	143	50
Chloride (g L^−1)	17.08	16.17	16.21	16.23
Hypochlorite (mg L^−1)	Nil	186	Nil	Nil
TDS (mg L^−1)	33.01	32.06	31.64	52.72
COD (mg L^−1)	7300 ± 0.10	4326	4294	292
BOD (mg L^−1)	4800	1900	1850	138

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3.1 Electro-oxidation of soak liquor

The electro-oxidation of soak liquor was done at a current density of 0.012 A cm⁻² for 30 minutes to reduce the cost of the process using a TSIA electrode. The oxidation of active chlorine molecules reduces the total energy required for the treatment.²¹ The impact of pH on electro-oxidation of soak liquor was studied by varying pH from 2 to 12 with an increment of 2. The amount of hypochlorite produced during the course of electro-oxidation at pH 2, 4, 6, 8, 10, and 12 was 401.8 ppm, 446.4 ppm, 483.6 ppm, 111.6 ppm, 74.4 ppm and 37.2 ppm respectively. The reduction in organic load was higher at pH 6 when compared to other pH values, where the COD removal efficiency was 41%. Under acidic conditions, the OCl⁻ ions are unstable and form HOCl as well as chlorine gas which are strong oxidants.

HOCl ↔ H⁺OCl⁻

The reduction in the concentration of hypochlorite was noticed at high pH values (pH 8–12).¹⁰ It can be concluded that near neutral pH is favourable for electro-oxidation of soak liquor with a higher quantity of hypochlorite production.¹⁰

In the electrochemical cell, chlorine formed at the anode and hydroxides formed at the cathode, which react to form chlorine and hypochlorites respectively.^10,19 Both the hypochlorite and free chlorine are chemically reactive and oxidize the organic pollutants in the effluent to carbon dioxide and water. The following reactions take place during electro-oxidation in the presence of sodium chloride.

At the anode:

2Cl⁻ → Cl₂ + 2e⁻ (1)

4OH⁻ → O₂ + 2H₂O + 4e⁻ (2)

At the cathode:

2H₂O + 2e⁻ → H₂ + 2OH⁻ (3)

Cl₂ + H₂O → H⁺ + Cl⁻ + HOCl (4)

The HOCl further dissociates into OCl⁻ and H⁺:

HOCl ↔ H⁺ + OCl⁻ (5)

Hypochlorite ions act as the main oxidizing agent in organic degradation.

The overall desired reaction of electrolysis is:¹²

Organic matter + OCl⁻ → intermediates + CO₂ + Cl⁻ + H₂O (6)

These oxidizing species can diffuse into the areas away from electrodes and continue to oxidize the pollutants.¹⁰ The UV absorbance spectra for electro-oxidized soak liquor at various pH values and commercial grade humic acid is presented in Fig. 2. It can be assumed that the presence of humic acid at pH 6 was due to efficient oxidation of organics present in soak liquor and the release of humic acid bound to it. The free humic acid could not be noticed at pH 2 and 4 though there is no significant variation in hypochlorite formation. It can be assumed that hypochlorite does not break the organics with humic acid significantly at low pH (2 to 4). This is because the organics can be removed from humic acid at pH 5.5 to 6.5 and also the humic acid complex is stable in the range of 3 to 5.²² Hence, the optimum pH for electro-oxidation to break the humic acid present in the soak liquor was about 6.

In the present study, the anode potential was in the range of 1.32–1.7 V vs. SCE. Salazar-Gastélum et al.²³ noticed the formation of hypochlorite at 1.7 V vs. SCE. In the present study, the oxidation of the organic complex is due to indirect electro-oxidation at the TSIA electrode.²⁴ The presence of a high concentration of sodium chloride (17.08 g L⁻¹) in soak liquor makes it even more compatible for the TSIA electrode with a current density of 0.012 A cm⁻² which is necessary for indirect oxidation.²³ Sundarapandiyan et al.¹⁰ performed electro-oxidation of tannery saline wastewater for 120 minutes by employing a graphite electrode, where the COD removal efficiency at a current density of 0.012 A cm⁻² was only about 89.11%. In the present study, the electro-oxidation time of soak liquor was reduced to 30 minutes employing TSIA electrodes thus, the energy consumption can be reduced which helped to overcome the shortcomings of the work done by the previous group.¹⁰ The reduction of time for electro-oxidation will enhance the life of the electrode thereby the cost can be reduced. In addition, 6 cycles of electro-oxidation were done and there was no coating damage and fouling on the electrodes which was confirmed by SEM and EDAX (ESI Fig. 1 and 2 and Table 1†). Kanagasabi et al.⁸ described that lower a COD concentration results in efficient degradation by microbes, which supports the present study.

3.2 Electro-biodegradation of soak liquor

Solar treatment after electro-oxidation helped in the removal of the oxidants generated during the electrochemical process.²⁸ The complete removal of hypochlorite from electro-oxidized soak liquor was achieved by exposing it to 1.0 × 10⁵ lux solar light for 1 hour for complete removal of hypochlorite followed by biodegradation.²⁵ The biological process was enhanced by an increase in biomass concentration which consequently improves the degradation efficiency.⁸ Thus supplementing the medium with a simpler substrate as the primary carbon source such as glucose increased the biodegradation efficiency.^8,26 Thus, the organics present in the solution were co-metabolised along with the primary carbon source. Two bacterial strains were isolated from the soak liquor and identified as Bacillus cereus and Klebsiella oxytoca. The phylogenetic tree was constructed and included in ESI Fig. 3.† The electro-oxidized soak liquor was treated using the bacterial strains isolated from soak liquor as a mixed culture after removal of hypochlorite by solar exposure.²⁷ The bacterial growth conditions were optimized and presented in Table 3. The growth curve of the mixed culture and individual isolates are mentioned in Fig. 1. It was indicated in earlier reports that microbes in the hypersaline environment have greater potential in degrading pollutants.^12,28 In the present study, the chloride concentration was about 17.08 g L⁻¹. Hence, these isolates can be claimed as halotolerant.^29,30

Table 3 Optimized parameters for the growth of a mixed culture for efficient biodegradation

Parameters	Optimized conditions for efficient biodegradation
Time of electro-oxidation	30 minutes
Primary carbon source	Glucose
Percentage of glucose	1%
pH	6
Percentage of inoculum	5%
Temperature	28 °C
Agitation	150 rpm

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Fig. 1 Growth curve of mixed and individual isolates comprising Bacillus cereus and Klebsiella oxytoca.

Fig. 2 UV-Vis spectra of soak liquor electro-oxidized at different pH values (2–4) with soak liquor and humic acid.

3.3 Chemical characterisation

A UV-Vis study was carried out for soak liquor before and after electro-oxidation and electro-biodegradation and is presented in Fig. 3. Two major peaks were observed at 197 nm and 200 nm, with an additional hump in the range of 252 to 294 nm. The presence of all peaks and a shoulder in the region of 250–270 nm denote humic acid.²⁹ After electro-oxidation of soak liquor, only one major peak was found at 210 nm which indicates the presence of hypochlorite. The peak at 210 nm corresponds to a π⋯π* electronic transition of carboxylic and phenolic groups.¹ Upon treatment of the electro-oxidized solution using microbes, the intensity of the peak at 210 nm further reduced significantly. The shifting of the peak from 210 to 230 nm in the electro-biodegraded and biodegraded sample is due to secondary metabolites of bacteria.

Fig. 3 UV-visible spectrum of humic acid, soak liquor before and after electro-oxidation, biodegradation and electro-biodegradation.

The FT-IR spectrum of soak liquor before and after electro-oxidation and electro biodegradation along with commercial grade humic acid is given in Fig. 4. The FT-IR spectrum of soak liquor is similar to commercial grade humic acid, which also confirms the presence of humic acid. It can be noticed that there is a significant decrease in humic acid bound with primary amine R–NH₂ (3440 cm⁻¹) after electro-oxidation, which is oxidized by OCl⁻ and converted to CO–NHR (1648 cm⁻¹). Another interesting observation is an increase in the intensity of peaks in the 3000 cm⁻¹ to 3150 cm⁻¹ region due to a breakdown of the humic acid organic complex. Tatzber et al.³¹ reported that humic acids are always involved in the formation of complexes with sodium salts of phenols to form sodium carboxylate salts (1700 cm⁻¹). It reveals that the humic acid complex is broken during electro-oxidation and in the biological treatment of soak liquor major peaks (OH, –NH₃⁺, –NH₂⁺, –CO–NH₂, –CO–NH, S–H, and P–H) could not be noticed. Humic acid exists in the form of sphere colloids, a rigid molecule,²⁹ which was broken down into smaller molecules during electro-oxidation by active oxidizing species and further used for biodegradation. The electro-biodegradation of soak liquor helped to break the rigid molecules, which can be observed through the intensity of peaks. The implementation of biological treatment after electrochemical oxidation has helped to oxidize the organic contaminants completely (Fig. 4) from the soak liquor.

Fig. 4 FT-IR spectrum of soak liquor. (A) Soak liquor, (B) after electro-oxidation, (C) after the biological process, (D) after electro-biodegradation, and (E) humic acid.

The CHNS analysis was carried out to measure the percentage of the respective elements present in the soak liquor before and after electro-oxidation and electro-biodegradation (ESI Fig. 4†). After electro-oxidation of soak liquor, no significant reduction of hydrogen was found, whereas the nitrogen and sulphur content reduced by 46% and 76% respectively. During electro-biodegradation of soak liquor, the removal efficiency of hydrogen, nitrogen and sulphur was 70%, 100%, and 84% respectively which indicates that electro-biodegradation increased the removal efficiency of the elements.¹⁹ On the other hand, in the stand-alone process of biological treatment, the removal efficiency of nitrogen and hydrogen was 50% and 60% respectively but the sulphur removal was similar to electro-biodegradation. These results support electro-biodegradation which is efficient in treating the soak liquor.

The pollution parameters COD, BOD and TKN were measured before and after electro-oxidation and electro biodegradation (Fig. 5A). After electro-oxidation, 60%, 36% and 64% of BOD, COD, and TKN reduced within 30 minutes respectively. After electro-biodegradation, the above parameters further reduced to 85%, 95%, and 88% respectively which supports the FTIR analysis. In the present study, the increased COD removal efficiency (95%) is due to electro-biodegradation and the application of mixed halophilic bacterial strains used in the study.¹² It can be claimed that the degradation efficiency is higher when compared with the previous studies.^6,8

Fig. 5 (A) Estimation of BOD, COD and TKN. (B) Estimation of protein and lipid in soak liquor before and after electro-oxidation and electro-biodegradation.

The protein and lipid content of the soak liquor was measured before and after electro-oxidation and electro-biodegradation (Fig. 5B). After electro-oxidation a reduction of 7.6% and 31% in protein and lipid was found, whereas after electro-biodegradation the above values further increased by 57% and 91% respectively. The biological treatment of soak liquor led to a poor reduction in protein and lipid; the reduction efficiency was 14% and 27%. The lower reduction of protein (7.6%) during electro-oxidation is due to the fact that protein was not completely mineralized but instead was broken down into simpler molecules such as smaller peptides and amino acids.¹⁰ The microbes were able to mineralize the amino acids only after the molecular breakdown of the humic acid organic complex.² This is the reason for the lower reduction in organic load during biodegradation alone when compared to electro-biodegradation. It can be concluded that electro-biodegradation gives better efficiency in the treatment of soak liquor within 7 days.

The soak liquor was analysed by HPLC before and after electrochemical and biological treatment and electro-biodegradation (Fig. 6). The untreated soak liquor has three peaks at retention times around 2.85, 3.161 and 5.482 min. After electro-oxidation, the peak intensity was reduced and the peak was found with retention times around 2.898 and 3.080 min. A new peak was observed at 5.471 min which is due to the molecular breakdown of the humic acid organic complex. After biological treatment of electrolysed soak liquor, the chromatogram showed six important peaks at retention times around 1.507, 2.703, 3.056, 3.181, 3.348, and 5.482 where the original peaks in the effluent disappeared; the formation of new peaks can be explained as metabolites of bacteria.¹⁹ During electro-biodegradation, the peak at 2.740 and 3.041 observed in soak liquor decreased by about 75.92% and 87.58% respectively. In the biodegradation process, a 29.67% and 22.71% reduction was observed respectively. These results also support that electro-biodegradation promotes significant degradation of organics present in the soak liquor.

Fig. 6 HPLC analysis of soak liquor, after biodegradation, electro-oxidation and after electro-biodegradation.

3.4 Mechanism proposed for electro-biodegradation of soak liquor

Considering the results from FT-IR, a possible mechanism can be proposed for the present study (Fig. 7). Humic acid bound with the primary amine R–NH₂ (3440 cm⁻¹) is oxidized by OCl⁻ and converted to CO–NHR (1648 cm⁻¹). It can be assumed that small degraded molecules formed after electro-oxidation [5-cyano-2,3,3′,4′-tetrahydroxy biphenyl, 2-(4-hydroxyphenyl)-3H-indole-4,7-dione derivative, 10H-phenoxazine-1,4-dione derivative, and 1,6,7-trihydroxyphenanthro[2,3]benzofuran-9,10-dione derivative (supported by FT-IR results)] are consumed by bacteria. CHNS analysis also revealed that 70% of hydrogen, 100% of nitrogen and 84% of sulphur were consumed by bacteria in the integrated process. Besides sulphur, the remaining counterparts of amino acids can be utilized by the bacteria. It can be concluded that electro-oxidation alone cannot be a complete treatment technique for soak liquor. Furthermore, it can be explained that the electro-oxidised hydrogen, nitrogen and sulphur molecules from amino acids can be easily consumed by bacteria.

Fig. 7 Possible mechanism of electro-biodegradation of soak liquor.

4 Conclusions

The electro-oxidation process has the ability to convert the biologically recalcitrant complex humic acids into biodegradable compounds. The smaller molecules present after electro-oxidation were effectively treated biologically using mixed cultures collected from tannery effluent. In this study, by applying a current density of 0.012 A cm⁻² for 30 minutes followed by biological degradation for seven days with primary substrate, an enhancement in the organic load reduction in soak liquor was found. The release of humic acid during electro-oxidation helped the biodegradation process. For the first time, the combined process achieved a higher COD reduction (95%) using halophilic bacteria at pH 6 for rich organic containing soak liquor. A higher cost efficiency of 0.03 dollars per cm³ subsequently reduces the cost by 97% and 90% when compared with the works of Sundarapandiyan et al.¹⁰ and Kanagasabi et al.⁸ The electro-biodegradation can be employed for effective treatment of soak liquor in the leather industry.

Acknowledgements

This work was supported by Council of Scientific and Industrial Research (CSIR), India under Zero Emission Research Initiative for Solid leather waste (ZERIS) Grant No. CSC0103.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra28076a

‡ The authors equally contributed to this work.

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