Biodegradation of Congo red dye in a moving bed biofilm reactor: Performance evaluation and kinetic modeling

Bioresource Technology 302 (2020) 122811

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Biodegradation of Congo red dye in a moving bed biofilm reactor: Performance evaluation and kinetic modeling

T

Ravi Kumar Sonwani, Ganesh Swain, Balendu Shekhar Giri, Ram Sharan Singh, ⁎ Birendra Nath Rai Department of Chemical Engineering & Technology, Indian Institute of Technology (BHU), Varanasi 221005, Uttar Pradesh, India

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Biodegradation Response surface methodology Congo red dye Moving bed biofilm reactor Elimination capacity

The biodegradation of Congo red dye was performed using polyurethane foam-polypropylene immobilized Bacillus sp. MH587030.1 in a moving bed biofilm reactor (MBBR). The central composite design (CCD) based response surface methodology (RSM) was used to optimize the process parameters; pH, Congo red concentration, and media filling ratio, and optimum conditions were observed to be 7.0, 50 mg/L, and 45%, respectively in batch MBBR. At optimum condition, MBBR was operated in continuous mode at different flow rates (25–100 mL/h) over a period of 564 h. The maximum removal efficiency (RE) and elimination capacity (EC) were obtained as 95.7% and 57.6 mg/L·day, respectively under steady-state. The kinetics of Congo red biodegradation at various flow rates were evaluated by a modified Stover-Kincannon model, and kinetic constants; KB and Umax were found to be 0.253 g/L·day and 0.263 g/L·day, respectively.

1. Introduction The extensive uses of synthetic dyes in various industries such as textiles, tanneries, carpets, cosmetics, and paper printing, discharge a wide range of complex effluents (Bharti et al., 2019; Chen et al., 2018). These effluents contain several harmful dyes that directly or indirectly affect the ecosystem (Hameed and Ismail, 2018). The discharge of untreated dyes directly into water bodies deteriorate the quality of water

⁎

(Shalini and Setty, 2019). According to Talha et al. (2018), more than 280,000 tons per annum of wastewater containing dyes are released into water bodies worldwide. The wastewater containing dyes is responsible for the reduction of dissolved oxygen, deterioration of photosynthesis, and obstruction of sunlight penetration (Koyani et al., 2013; Unnikrishnan et al., 2018). Amongst the different type of dyes, the azo dye has been broadly used in various industries and share about more than 60% of the total application of dyes (Ayed et al., 2010; Roy

Corresponding author. E-mail address: [email protected] (B.N. Rai).

https://doi.org/10.1016/j.biortech.2020.122811 Received 24 October 2019; Received in revised form 11 January 2020; Accepted 13 January 2020 Available online 21 January 2020 0960-8524/ © 2020 Elsevier Ltd. All rights reserved.

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has been given on the biodegradation of dye using PUF-PP as the moving carriers in MBBRs. Also, limited efforts have been applied to improve the efficiency of MBBR using optimization techniques such as CCD of RSM for the removal of dye. The present work focused on the application of MBBR for the effective biodegradation of dye using polyurethane foam-polypropylene immobilized Bacillus sp. MH587030.1. In this study, Congo red (CR) was selected as a model azo dye. The process parameters such as carrier filling ratio, CR dye concentration, and pH were optimized in MBBR using CCD of RSM. The performance of a continuous MBBR was evaluated at different flow rates under optimized conditions. Furthermore, dye degradation kinetics in MBBR was evaluated by a modified Stover–Kincannon model.

et al., 2018). Furthermore, the azo dyes and their intermediates are precarious, carcinogenic, and mutagenic in nature, which leads harmful effects on humans and aquatics life (Chen et al., 2018). The United States of Environmental Protection Agency (US EPA) has also listed these dye as hazardous pollutants (Papadopoulou et al., 2013). Congo Red (CR) [1-naphthalene sulfonic acid, 3,3′-(4,4′-biphenylenebis (azo)) bis(4-amino-) disodium salt] is a type of azo dye, which is extensively used in textile dying industry (Talha et al., 2018). It is toxic to animals and plants and can causes carcinogenic and mutagenic effects on humans and aquatics life (Lade et al., 2015). It is very challenging to remove the dyes from wastewater due to their high stability (Shalini and Setty, 2019; Lade et al., 2015). The wastewater containing azo dye is a major challenge to the researchers (Bharti et al., 2019). Therefore, extensive efforts are needed to develop an economical, ecofriendly, and advance technique to overcome the concern of wastewater containing dyes. In this direction, several conventional methods such as adsorption, electrolysis, ozonation, coagulation/precipitation, and UV-Fenton oxidation have been used for the removal of dyes from wastewater (Bharti et al., 2019; Hameed and Ismail, 2018; Koyani et al., 2013). However, high sludge generation, transfer of pollutants from one phase to another (e.g., adsorption), and toxic by-products are demerits of existing conventional treatment methods (Vikrant et al., 2018; Shalini and Setty, 2019). The biodegradation of dyes has been reported as a realistic option to (Bankole et al., 2019; Bharti et al., 2019). It is also a cost-effective and environmentally benign technique that completely breakdown or mineralize the pollutants into simpler products (Roy et al., 2018; Vikrant et al., 2018; Sonwani et al., 2019b). Previously, several microorganisms such as Pseudomonas aeruginosa, Shewanella oneidensis, Aeromonas hydrophila, Alcaligenes faecalis, Acinetobactor baumannii, Klebsiella quasipneumoniae, Bacillus sp., Aeromonas sp., etc. have been applied for the biodegradation of dyes (Bharti et al., 2019; Kashefi et al., 2019; Srinivasan and Sadasivam, 2018). Despite the merits of biodegradation, it is challenging to select the efficient microorganisms for the degradation of specific pollutants such as azo dyes at high loading rate. Enormous efforts have been given to enrich the microorganism from specific dye contaminated sites to enhance the efficacy of the biodegradation process (Chen et al., 2018). Generally, two types of biodegradation system namely free cell in which microorganism directly added into bioreactor, and immobilized cell in which microorganism grown on carrier surface or encapsulated into carriers have been largely used by the previous researchers to biodegrade the dyes in wastewater (Padmanaban et al., 2015; Shalini and Setty, 2019). Microorganism immobilized on packing support shows better degradation potential towards dye as compared to free cells (Vikrant et al., 2018). According to Padmanaban et al. (2015), immobilized or encapsulated microorganism shows improved RE, better control of the process, and high cell-mass retention during biodegradation. Bharti et al. (2019) have also reported that the microorganism immobilized into bio-char shows enhanced RE towards methylene blue as compared to the free cells. For the application of immobilized microorganisms, various bioreactors such as packed bed bioreactors (PBBR), rotating biological contactors (RBC), tricking biofilters (TBF), moving bed biofilm reactors (MBBR), and other bioreactors have been widely studied for the biodegradation of dyes (Bharti et al., 2019; Leyva-Díaz et al., 2013; Vikrant et al., 2018; Shalini and Setty, 2019). However, MBBR is considered as the most effective bioreactor in terms of high loading rate, effective mass transfer, and small space requirements (Oberoi and Philip, 2017). Additionally, in MBBR, the biodegradation of pollutants is encounter by both suspended as well as attached biofilm (Leyva-Díaz et al., 2013). Recently, MBBR has been extensively tested for the biodegradation of the wide range of pollutants (Derakhshan et al., 2018a; Deng et al., 2016). Sonwani et al., (2019a) studied the biodegradation of polycyclic aromatic hydrocarbon in three MBBRs and revels that PUF-PP immobilized microorganisms show better RE. However, minimal attention

2. Materials and methods 2.1. Dye and chemicals Congo red (CAS number 573-58-0) is a type of azo dye was purchased from Sigma-Aldrich, India. The mineral salt media (MSM) and other chemicals were purchased from Merck, India. The modified MSM (g/L) contains the following composition; KH2PO4 (1.8), NaH2PO4 (2.5), FeCl3 (0.01), MnSO4·H2O (0.2), MgSO4 (0.2), beef extract (2.0), peptone (1.0), and supplemented with 0.2% (w/v) of glucose (Chen et al., 2018). The synthetic wastewater was prepared with different concentrations of CR dye in MSM. The pH of the medium was adjusted with adding 0.1 N NaOH/0.1 N H2SO4.

2.2. Enrichment of dye degrading bacterial culture and molecular characterization The textile industries located at Bhadohi, India, have been disposing of the waste effluents which contain different types of dyes. This site was selected for the isolation of potential bacterial species due to the possibility of dye degrading microorganisms present in the deposited soil sample. The soil samples were collected in sterile polyethylene bags, and until further use, the samples were kept at 4 ⁰C. The isolation and enrichment procedure of bacterial species from soil samples has been described by Sonwani et al. (2018). In brief, initially, the soil sample was enriched with MSM (100 mL) and Congo red dye (20 mg/L) in Erlenmeyer flasks (250 mL) and incubated at 120 rpm and 37 °C for five days. After completion of the incubation period, 2 mL of inoculums was transferred to another Erlenmeyer flasks containing fresh 100 mL of MSM and Congo red (50 mg/L) and further acclimatized until entire dye degradation observed under similar state. This process was repeated thrice with gradually increasing the Congo red dye. A serial dilution technique was applied to isolate the bacterial species. Based on batch experiments, most potential bacterial sp. was selected and used in this study. The molecular characterization of potential bacterial species was carried out at Triyat Scientific, Nagpur, India. The conventional CTAB (Cetyl trimethyl ammonium bromide) method was used to purify the DNA. The DNA was PCR (polymerase chain reaction) amplified using the 16S-rRNA forward (27F-5′ AGAGTTTGATCMTGGCTCAG3′) and reverse (1492R-5′ TACGGYTACCTTGTTACGACTT3′) primers. PCR was run for 30 cycles under the following conditions: denaturation at 94 °C for 30 sec, annealing at 60 °C for 30 sec, and extension at 72 °C for 1 min. The obtained PCR sample was sequenced by ABI 3730xl sequencer (Applied Biosystems). The obtained 16S-rRNA sequence was submitted in the GenBank database (NCBI) and compared with those available in the database using nucleotide BLAST algorithm. The sequence was aligned with MUSCLE alignment tool, and evolutionary study were performed using the neighbor-joining (NJ) methods of MEGA 6.0 software. 2

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Fig. 1. Schematic representation of experimental setup for the biodegradation of Congo red dye. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.3. Experimental set-up

Table 2 Central composite design of experimental runs and their outcomes for the removal of Congo red dye.

The experimental set up was made of a wastewater tank, MBBR, air pump (KNF, Labport, Germany), rotameter (SRS-MG5, Eureka, Pune), and peristaltic pump (Miclins PP 30 EX) (Fig. 1). MBBR bioreactor was fabricated of borosilicate glass with 2.0 L of working capacity. PUF-PP was used as moving carriers in MBBR due to its highly porous, excellent mechanical strength, low-cost, and reusable nature (Shalini and Setty, 2019; Sonwani et al., 2019a). The porous structure of carriers facilitates effective microbial growth and colonization. The details and preparation procedure of moving carriers (PUF-PP) were described by Sonwani et al. (2019a). The average weight of each PUF-PP carrier was 1.13 ± 0.04 g. The sterilized air (passed through 0.22 µm filter) was continuously supplied in MBBR to keep the circulation of carriers and prevent from ambient contamination. Initially, the culture of dye degrading bacterial species was inoculated in MBBRs containing glucose and run for ten days for biofilm development (Deng et al., 2016). Three identical MBBR was operated in parallel for the effective interpretation of data. 2.4. Optimization by response surface methodology (RSM) The biodegradation of CR dye in MBBR was optimized by CCD of RSM. In this technique, the interactive effects of process variables such as carrier filling ratio, pH, pollutant concentration, temperature, and hydraulic retention time have been widely examined (Álvarez et al., 2015; Sonwani et al., 2019c). It also offers a greater yield with less effort than one variable at a time (OVAT) technique for the optimization of process variables (Dubey et al., 2016; Gusain et al., 2016). For this, a design Expert 11 software (Stat-Ease Inc., Minneapolis, USA) has been widely applied (Dubey et al., 2016; Sutar et al., 2019). In the present study, the process variables such as pH (5.0–9.0), dye concentration (10–100 mg/L), and carrier filling ratio (10–60%) were selected to optimize the biodegradation of CR dye in MBBR (Table 1). The total runs were designed according the following term2n + 2n + n 0

Name

Unit

Minimum

Maximum

Mean

A B C

pH Dye concentration Carrier filling ratio (%)

– mg/L –

5 10 10

9 100 60

7 55 35

pH

Dye conc. (mg/L)

Carrier filling ration (%)

Dye removal (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

7 7 5 5 7 5 9 9 7 5 9 7 7 5 9 7 9 7 7 7

55 55 100 10 55 10 10 100 55 55 55 55 55 100 10 10 100 55 55 100

35 35 10 10 10 60 60 60 35 35 35 35 35 60 10 35 10 35 60 35

90 92 33 56 69 63 72 38 92 62 69 89 93 44 52 99 17 89 97 76

using CCD of RSM. Where n and no represent the number of independent variables and repetitions of experiments at the center point, respectively. A total of 20 experiments were performed, and their outcomes in terms of dye RE (%) are given in Table 2. In order to predict the relevant model, a second-order polynomial equation was applied (Eq. (1)). Y = β0 + βi Xi + βj Xj + βij Xij + βii Xi 2 + βjj Xj 2 +……… (1)where Y is dye removal efficiency (%), β is the correlation coefficient, and i and j are the coefficients of multi-degree. The analysis of variance (ANOVA) was applied to experimental data to find out the adequacy of different models, namely linear, interaction, and quadratic. Based on the ANOVA analysis, the most suitable model was evaluated via values of R2 and adjusted R2adj. The p-value greater than 0.05 designates that the model terms are significant, while the pvalue greater than 0.1 shows insignificant terms (Kashefi et al., 2019; Silva et al., 2018).

Table 1 The range of the operating parameters in the biodegradation of Congo red dye. Factor

Run

2.5. Continuous study in a moving bed biofilm reactor The performance of a continuous MBBR at different flow rates was 3

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V KB V 1 = + Q (So − Se ) Umax QSo Umax

examined under optimum conditions obtained in the batch study. The dye solution (wastewater) was stored in 50 L plastic tank. The dye solution (50 mg/L) was continuously supplied into MBBR at varying flow rates of 25–100 mL/h using a peristaltic pump. At every flow rate, adequate time was given to achieve the steady-state conditions. All the analyses were carried in triplicate, and their average value was used for the data interpretation. The MBBR was continuously was operated for 564 h, and its performance can be estimated by following equations (Geed et al., 2017; Yadav et al., 2014):

%Removalefficiency(RE) =

Sin − Sout × 100 Sin

(Sin − Sout ) × Q Eliminationcapacity(EC) = V

Sin × Q Inletloadingrate(IL) = V

(6)

The mass balance of the substrate is given by following equation:

dS QS0 = QSe + V ⎛ ⎞ ⎝ dt ⎠

(7)

Solving Eq. (5) and (7) results in Eq. (8)

QSo = QSe + (2)

QSo ) V QSo (V )

Umax ( KB +

×V (8)

Furthermore, Eq. (8) can be solved to predict the outlet concentration of dye (Eq. (9)) and volume required of MBBR (Eq. (10)).

(3)

Se = So − (4)

V=

where Sin and Sout is the inlet and outlet concentration of Congo red dye in MBBR, V is the active volume of the MBBR, and Q is the volumetric flow rate.

Umax So KB + (

QSo ) V

(9)

QSo

(

Umax So So − Se

)−K

B

(10)

3. Results and discussion 2.6. Analytical techniques 3.1. Identification of dye degrading bacterial species The residual concentration of Congo red dye was analyzed by a UV–vis spectrophotometer (ELICO SL-2010). Before analysis, the treated samples were centrifuged at 5000×g for 10 min, and the supernatant was used to measure the dye concentration. The morphology of biofilm developed onto MBBR carriers was analyzed by a SEM (EVO18, ZESIS). Before SEM analysis, the MBBR carriers were vacuum dried (NSW-251, India), and followed by spray coating with gold particles to attain the nominal resolution. The PUF-PP carriers without bacterial cells were used as the control (0th day). The pH was measured using a pH meter (HD 2305.0; Delta OHM; Italy). The metabolites of the Congo red degraded sample were analyzed GC–MS (QP201 Shimadzu, USA). For this, the treated sample was centrifuged at 5000×g for 10 min to remove the suspended biomass, and supernatant was twice extracted with an equal volume of ethyl acetate. Further, the sample was filtered through 0.22 µm pore size filter and analyzed by GC–MS. The functional group of Congo red dye and its biodegraded samples were analyzed by FTIR analysis (NICOLET 5700, Japan).

The obtained 16S-rRNA sequence of dye degrading bacterial sp. was deposited in the GenBank database of NCBI and get the accession number (MH587030). The sequence was organized with maximum similarity, and five sequences were selected and exported in FASTA format. The sequence shows more similarity with Bacillus sp. (MH587030.1). The details of obtained sequence and constructed a phylogenetic tree of Bacillus sp. (MH587030.1) have been described in E-Supplementary data. 3.2. Morphological analysis of packing media The surface morphology of the PUF-PP carrier was analyzed by SEM. The morphology of PUF-PP before (0th day) and after (15th day) biofilm formation is given in E-Supplementary data. A large number of micropores connected to each other was observed on the PUF-PP surface before the formation of biofilm. These micropores offer an enormous surface area for biofilm formation (Geed et al., 2018). The dense bacterial biofilm is formed into the surface and pores of PUF-PP and reveals the successful colonization of bacterial cells on carriers. Further, the biofilm developed system was used for the biodegradation of the Congo red dye. The formation of biofilm into carriers is due to the accumulation of active biomass. The process variables namely; pH, temperature, pollutant concentration, and effluent flow rates affect the biofilm formation (Sonwani et al., 2019d). The extracellular polymers released by microorganisms support the development of stable biofilm on carrier against the high hydraulic load (Derakhshan et al., 2018b). The Bacillus cohnii immobilized into PUF was used for the biodegradation of Reactive Red 120 dye in wastewater (Padmanaban et al., 2015). They reported that PUF provides a highly porous surface for the formation of stable biofilm. Similarly, Kureel et al., (2017) used PUF as the packing support for the formation of biofilm and reported that immobilized PUF-Bacillus sp. successfully degrade the benzene at the high concentration.

2.7. Kinetics of dye degradation Mathematical and kinetic models are helpful in the design and modeling of bioreactors. These models are not only important at laboratory scales but also supportive in the design of the industrial process (Derakhshan et al., 2018a). Several kinetic models have been developed and successfully tested in batch and continuous operations (Sonwani et al., 2019d; Geed et al., 2017; Derakhshan et al., 2018a; Singh et al., 2010). In this direction, a modified Stover–Kincannon model is widely applied for evaluating the kinetic constant of MBBR bioreactor for the biodegradation of pollutants (Ahmadi et al., 2017; Kapdan, 2005). Therefore, Modified Stover–Kincannon model was applied to evaluate the kinetics of dye degradation, and its simple form can be presented by Eq. (5) (Ahmadi et al., 2017; Sonwani et al., 2019a). QS )

Umax ( Vo dS Q (So − Se ) = = QS dt V KB + ( V o )

3.3. Statistical analysis and optimization

(5)

where S0 and Se represent the initial and final concentrations of dye (mg/L), respectively. Q, V, KB, and Umax are the inlet flow rate (L/day), volume of the reactor (L), saturation rate constant (mg/L·day), and maximum dye removal rate (mg/L·day), respectively. The linear form of the Eq. (6) is given as.

3.3.1. Statistical analysis The obtained the data were examined with linear, interaction, and quadratic models, and the obtained table is given in E-Supplementary data. The values of R2 and adjusted R2 were obtained as 0.98 and 0.96, respectively for the quadratic model. Furthermore, the value of 4

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microorganisms and considered as an essential variable in biodegradation of dye (Chen et al., 2018). The maximum RE of dye was obtained at 7.0 pH. As we change the pH either acidic or alkaline at fixed carrier ratio, the RE of the system dramatically declined. The bacteria produce enzymes having ionic group on their active site, and these ionic group must be in the suitable state (acid or base) for effective biodegradation of CR dye. The activity of ionic form of active site is changed due to variation of pH, which adversely affects the activity of the enzyme and hence RE. At neutral pH, the dye RE was significantly enhanced by increasing the carrier filling ratio (%). However, above 35% of carrier filling ratio, very less enhancement in dye RE was observed. The increased in the dye RE with carrier filling ration may be due to significant amount of biofilm developed at higher carrier filling (%) in MBBR. However, when more carrier filling ratio was increased, the carriers were densely packed with limited void space in the bioreactor and making it tough to move freely. With increasing more carriers filling ratio, a thick layer of biofilm developed around the carriers, which leads inefficient diffusion of oxygen and substrate inside the developed biofilm and hence affects the RE (Lopez-Lopez et al., 2012; Barwal and Chaudhary, 2015).

Table 3 ANOVA summary for the quadratic model. Source

Sum of Squares

df

Mean Square

F-value

p-value

Model A-pH B-Conc. C-Carrier filling ratio AB AC BC A2 B2 C2 Residual Lack of Fit Pure Error Cor Total

10568.69 10.00 1795.60 756.90

9 1 1 1

1174.30 10.00 1795.60 756.90

69.03 0.5879 105.56 44.50

< 0.0001 0.4610 < 0.0001 < 0.0001

91.13 66.13 3.13 2163.01 100.51 305.82 170.11 155.27 14.83 10738.80

1 1 1 1 1 1 10 5 5 19

91.13 66.13 3.13 2163.01 100.51 305.82 17.01 31.05 2.97

5.36 3.89 0.1837 127.16 5.91 17.98

0.0432 0.0769 0.6773 < 0.0001 0.0354 0.0017

10.47

0.0111

significant

significant

predicted R2 (0.89) is in a reasonable covenant with adjusted R2 (0.96) and seems to be well fitted with results. The obtained values of R2, adjusted R2, and predicted R2 are reasonable with previous studies and suggest that the quadratic model is suitable for optimization study (Sonwani et al., 2019a; Gusain et al., 2016). The experimental data fitted with the quadratic model is given by following Eq. (11):

3.3.2.3. Interactive effect of initial dye concentration and carrier filling ratio. Fig. 2 e and f demonstrate the simultaneous effect of initial dye concentration and carrier filling ratio on the dye removal. The dye RE was enhanced with carrier filling ratio, whereas the inverse relationship was observed against dye concentration. For example, the dye RE of 69% was observed with 10% carrier filling ratio, while RE increased with carrier filling ratio and reached 89% with 35% of carrier filling at 55 mg/L of dye. These results intimated that an increase in carrier filling in MBBR causes more surface area was available for the biofilm formation; hence, more active and stable biomass formation takes place into MBBR carriers. The decrease in the RE at high concentration CR dye was due to substrate inhibition and toxicity of dye towards microorganisms (Talha et al., 2018). The high concentration of pollutants unfavorably inhibits the enzymatic activity of bacterial species and hence affects the biodegradation efficacy of the system (Bankole et al., 2018; Yadav et al., 2014). Padmanaban et al. (2015) have studied the degradation of dye using Bacillus cohnii in a bioreactor and reported that the efficacy of bioreactor becomes low due to substrate inhibition at higher loading of dye.

Dyeremoval (%) = 91.9 − 1.0A − 13.4B + 8.7C − 3.4AB + 2.87AC + 0.63BC − 28.1 (11) A2 − 6.1B2 − 10.5C 2 ANOVA analysis was used to examine the significance of each variable for dye degradation and the summary of the result is reported in Table 3. In this work, B, C, AB, A2, B2, and C2 are significant model terms (p < 0.05). The obtained model F-value of 69.1 denotes that the overall model is suitable for dye degradation study. There is only a 0.01% error chance could occur due to noise. Therefore, the quadratic regression model was significant to correlate the response and independent variables for the optimization of the biodegradation system. 3.3.2. Optimization study using a central composite design 3.3.2.1. Interactive effect of pH and initial dye concentration. Fig. 2 shows the three-dimensional surface response and counter plots of the interactive variables namely; pH, initial dye concentration, and carrier filling ratio against the removal of Congo red dye in MBBR. The plots in Fig. 2a and b show the simultaneous effect of initial dye concentration and pH against the dye RE. The elliptical profile of a contour plot reveals a very significant interaction between dye concentration and pH. As we change the pH either acidic or alkaline, the dye RE in MBBR was decreased. At fixed carrier filling ratio (35%) and pH 7.0, the dye RE of 99% was achieved at 10 mg/L, whereas the RE decreased with increasing dye concentration and reached up to 76% at 100 mg/L (Table 2). Furthermore, at similar carrier filling ratio and initial dye concentration of 55 mg/L, the RE of 92% was observed at pH 7.0, while RE decreased to 62 and 69% at pH 5.0 and 9.0, respectively. The enzymatic activity of microorganisms is adversely affected in acidic and alkaline condition, and corresponding dye RE was significantly reduced (Gopinath et al., 2009; Nath et al., 2019; Sutar et al., 2019). The decrease in the RE of dye with increasing dye concentration was due to substrate (dye) inhibition (Talha et al., 2018; Padmanaban et al., 2015).

3.3.3. Validation of the model In order to obtain an optimal condition for the maximum removal of dye, CCD of RSM software was used. The optimal values of pH, dye concentration, and carrier filling ratio were predicted and found as 7.1, 50 mg/L, 45.2%, respectively. In the validation process, pH, dye concentration, and carrier filling ratio were rounded off to 7.0, 50 mg/L, and 45%, respectively. The obtained experimental response was in good agreement with predicted value and revealed only 1.57% of error with predicted results (E-Supplementary data). The error of < 5% indicates the suitability and validity of the model (Dubey et al., 2016). 3.4. Biodegradation of Congo red dye in a continuous bioreactor The effect of flow rates on the dye RE was studied to achieve a suitable flow rate at which the MBBR could offer high performance. The performance of continuous MBBR was evaluated at various flow rates (25–100 mL/h) under optimum conations (pH = 7.0, dye concentration = 50 mg/L, and carrier filling ratio = 45%). Initially, the MBBR was run at 25 mL/h with an ILR of 20 mg/L.day. The dye RE and EC were exponentially increased with time and reached equilibrium in 136 h with 95.7% of RE and 19.1 mg/L/day of EC (Fig. 3a). The overall performance of MBBR at different flow rates has given in Table 4. The inlet flow rate was increased from 25 to 50 mL/h on 144th h, and it was observed that a sharp dip in RE observed. The RE was further recovered (91.82%) with time and became almost constant in 240 h. At the same

3.3.2.2. Interactive effect of pH and carrier filling ratio. The plots in Fig. 2c and d represent the simultaneous effect of pH and initial dye concentration against the dye RE. The pH of the solution is responsible for the transport of dye molecules through the cell membrane of 5

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Fig. 2. Surface and contour plots for the removal (%) of Congo red dye: (a, b) effect of pH and dye concentration; (c, d) effect of carrier filling (%) and pH; (e, f) effect of carrier filling (%) and dye concentration. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

time, the EC was increased from 19.1 to 36.1 mg/L.day, as the flow rate was increased from 25 to 50 mL/h. On 175th h, the flow rate was increased to 75 mL/h, and it was found that after a sharp initial dip on RE in 264 h, the RE was reached to 88.9% on 396th h. Further, on 408th h, as we increased the flow rate from 75 to 100 mL/h, a sharp drop of RE was observed, which again stabilized in 564th h with 72.29% of RE. The maximum EC of 57.6 mg/L·day was found at 100 mL/h of inlet flow rate. The dye RE was decreased to 95.7, 91.8, 88.9, and 72.9% as the flow rates were increased to 25, 50, 75, and 100 mL/h, respectively. The high flow rate of dye solution could lead to short hydraulic retention time (HRT) inside MBBR, and corresponding RE was significantly reduced. It was reported that a sufficient HRT required for the effective degradation pollutants (Banerjee and Ghoshal, 2017; Leyva-Díaz et al., 2013; Sonwani et al., 2019a). The high inlet flow rates also impede the

effective growth of bacteria into the surface of carriers and reduce the interaction of dye molecules, thereby lees RE observed. A similar kind of work has been performed by the previous researcher and informed that the RE of pollutants was reduced due to short hydraulic retention time (HRT) at the high inlet flow rate (Geed et al., 2018; Kurade et al., 2017). Kurade et al. (2017) have studied the degradation of textile effluent at various flow rates by the bacterial-yeast consortium and found that as the inlet flow rate increased, the RE was extremely reduced. Similarly, Nath et al. (2016) studied the biodegradation of malachite green at different flow rates by immobilized calcium alginate Bacillus cereus and reported that the RE malachite green was decreased at the high inlet flow rate.

6

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Table 5 Profile of kinetics constants calculated during biodegradation of Congo red dye. Dye

Umax (g/L·day)

Kb (g/L·day)

R2

Congo red

0.263

0.253

0.995

mass transfer control was observed at low ILR, whereas bio-reaction control limitation was found at high inlet loading rate. It is uneconomical to operate the PBBR in the mass transfer limitation zone (Yadav et al., 2014; Geed et al., 2017). From the practical point of view, the desirable lower limit of ILR in the bioreactor at which the controlling mechanism changes from mass transfer control to bio-reaction control, whereas the point of intersection of RE and EC may be considered as extreme limit (Geed et al., 2017). In this work, the optimum operating range of the inlet loading rate has been obtained between 60 and 72.3 mg/L·day for the effective degradation of Congo red dye. 3.6. Kinetic study by modified Stover-Kincannon model In this work, the kinetics of Congo red dye degradation in an MBBR was studied using a modified Stover-Kincannon model. A graph between reciprocal of total organic loading rate [V/(Q × S0)] vs. reciprocal of total organic loading removal rate [V/(Q(S0−Se)] is given in E-Supplementary data. The values of saturation constant (KB) and maximum dye removal rate (Umax) were determined as 0.253 and 0.263 g/L·day, respectively from slope and intercept. The summary of kinetic constants is given in Table 5. The value of R2 (0.99) indicates that the model is well fitted with experimental data. Kapdan (2005) studied the biodegradation kinetics of dyestuff (Reactive red 195) using a modified Stover-Kincannon model and kinetic constants: KB and Umax were reported as 0.43 g/L·day and 0.47 g/L·day, respectively. The naphthalene biodegradation kinetics was studied by a modified StoverKincannon model, and kinetic constants were observed as KB: 0.769 g/ L·day and Umax: 0.87 g/L·day (Sonwani et al., 2019a). In this work, the obtained values of KB and Umax are smaller than those reported by Kapdan (2005) and Sonwani et al. (2019a). The variation of the kinetic parameter (KB and Umax) with previous work may be due to distinct in the composition and inlet loading rate of target pollutants, types of bacterial species used, operating process parameters, and design of bioreactor. The model also suggests that the dye removal rate is adversely affected by ILR. Furthermore, by putting the values of kinetic constants in Eqs. (9) and (10), the dye effluent concentration and the required volume of MBBR can predict using Eqs. (12) and (13), respectively.

Fig. 3. Performance of a continuous moving bed bioreactor: (a) Effect of inlet loading rate on removal efficiency and elimination capacity; (b) Relation between mass transfer and bio-reaction controlling zone at various inlet loading rates. Table 4 Performance of a moving bed bioreactor for the biodegradation of Congo red dye. Flow rate (mL/h)

Process time (h)

ILR (mg/L·day)

EC (mg/L·day)

RE (%)

25 50 75 100

1–132 144–240 252–396 408–564

20 40 60 80

19.1 36.1 52.0 57.6

95.7 91.8 88.9 72.2

ILR = Inlet loading rate; EC = Elimination capacity; RE = Removal Efficiency.

3.5. Rate control mechanism in dye degradation The effect of ILR of dye solution on the RE and EC is shown in Fig. 3b. As the ILR of dye solution was increased, the RE was slightly decreased up to 60 mg/L·day of ILR and beyond this, the RE was extremely declined. The linear relationship was found between the ILR and EC up to 60 mg/L·day of ILR, and thereafter, EC was increased very slowly with a non-linear pattern. The maximum EC of 57.67 mg/L·day was obtained at 80 mg/L·day of ILR. At a high flow rate, the performance of bioreactors was significantly decreased due to rate controlling mechanism (Chung et al., 2003; Yadav et al., 2014). In the present work, two distinct zones of mass transfer and bio-reaction control were observed during biodegradation of Congo red dye at different ILR. At low ILR, the diffusion of substrate molecules through biofilm was slow and lead to mass transfer limitations (Yadav et al., 2014; Kathiravan et al., 2014). Due to mass transport limitation, the internal layer of biofilm could be substrate deficient and the microorganisms did not utilize the substrate completely (Dizge and Tansel, 2010; Yadav et al., 2014). The biodegradation of the substrate was transformed from mass transfer to bio-reaction control due to high diffusional flux at high ILR (Yadav et al., 2014). Geed et al., (2018) studied the biodegradation of Malathion at different ILR using Bacillus sp. immobilized into PUF and reported that the

Se = So −

V=

0.263So 0.253 + (

QSo ) V

(12)

QSo

(

0.263So So − Se

) − 0.253

(13)

These results demonstrated that the modified Stover-Kincannon model could be used effectively for the biodegradation kinetics study of dye in MBBR and scale-up the process. 3.7. Biodegradation analysis. The spectral profile of the control and biodegraded sample of CR dye is represented in Fig. 4. The significant decrease in the absorbance of the CR degraded sample was observed and confirmed the degradation of dye in MBBR. The FTIR spectrum of the untreated (control) and treated sample of CR dye is reported in E-Supplementary data. The FTIR spectrum of untreated CR dye shows number of peaks at different wavenumber; a peak at 678 cm−1 due to bending and vibration of CeH, a peak at 863 cm−1 due to p-distributed ring vibration, a peak at 7

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Acknowledgments The author (RKS) thankfully acknowledges the Indian Institute of Technology (BHU) Varanasi, India, for providing laboratory facilities to perform the work. The author gratefully acknowledges the Ministry of Human Resource Development (MHRD), New Delhi, India, for providing the financial support. This work also acknowledges the support made by Scheme for Promotion of Academic and Research Collaboration (SPARC), India. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2020.122811. References Ahmadi, M., Jaafarzadeh, N., Rahmat, Z.G., Babaei, A.A., Alavi, N., Baboli, Z., Niri, M.V., 2017. Kinetic studies on the removal of phenol by MBBR from saline wastewater. J. Environ. Heal. Sci. Eng. 15, 1–7. Álvarez, M.S., Rodríguez, A., Sanromán, M.Á., Deive, F.J., 2015. Simultaneous biotreatment of Polycyclic Aromatic Hydrocarbons and dyes in a one-step bioreaction by an acclimated Pseudomonas strain. Bioresour. Technol. 198, 181–188. Ayed, L., Khelifi, E., Jannet, H. Ben, Miladi, H., Cheref, A., Achour, S., Bakhrouf, A., 2010. Response surface methodology for decolorization of azo dye methyl orange by bacterial consortium: produced enzymes and metabolites characterization. Chem. Eng. J. 165, 200–208. Banerjee, A., Ghoshal, A.K., 2017. Biodegradation of an actual petroleum wastewater in a packed bed reactor by an immobilized biomass of Bacillus cereus. J. Environ. Chem. Eng. 5, 1696–1702. Bankole, P.O., Adekunle, A.A., Govindwar, S.P., 2019. Demethylation and desulfonation of textile industry dye, Thiazole Yellow G by Aspergillus niger LAG. Biotechnol. Rep. 23, e00327. Bankole, P.O., Adekunle, A.A., Govindwar, S.P., 2018. Enhanced decolorization and biodegradation of acid red 88 dye by newly isolated fungus, Achaetomium strumarium. J. Environ. Chem. Eng. 6, 1589–1600. Barwal, A., Chaudhary, R., 2015. Impact of carrier filling ratio on oxygen uptake & transfer rate, volumetric oxygen transfer coefficient and energy saving potential in a lab-scale MBBR. J. Water Process. Eng. 8, 202–208. Bharti, V., Vikrant, K., Goswami, M., Tiwari, H., Sonwani, R.K., Lee, J., Tsang, D.C.W., Kim, K.H., Saeed, M., Kumar, S., Rai, B.N., Giri, B.S., Singh, R.S., 2019. Biodegradation of methylene blue dye in a batch and continuous mode using biochar as packing media. Environ. Res. 171, 356–364. Chen, Y., Feng, L., Li, H., Wang, Y., Chen, G., Zhang, Q., 2018. Biodegradation and detoxification of direct black G textile dye by a newly isolated thermophilic microflora. Bioresour. Technol. 250, 650–657. Chung, T.P., Tseng, H.Y., Juang, R.S., 2003. Mass transfer effect and intermediate detection for phenol degradation in immobilized Pseudomonas putida systems. Process Biochem. 38, 1497–1507. Deng, L., Guo, W., Ngo, H.H., Zhang, X., Wang, X.C., Zhang, Q., Chen, R., 2016. New functional biocarriers for enhancing the performance of a hybrid moving bed biofilm reactor-membrane bioreactor system. Bioresour. Technol. 208, 87–93. Derakhshan, Z., Ehrampoush, M.H., Mahvi, A.H., Dehghani, M., Faramarzian, M., Ghaneian, M.T., Mokhtari, M., Ebrahimi, A.A., Fallahzadeh, H., 2018a. Evaluation of a moving bed biofilm reactor for simultaneous atrazine, carbon and nutrients removal from aquatic environments: Modeling and optimization. J. Ind. Eng. Chem. 67, 219–230. Derakhshan, Z., Mahvi, A.H., Ehrampoush, M.H., Mazloomi, S.M., Faramarzian, M., Dehghani, M., Yousefinejad, S., Ghaneian, M.T., Abtahi, S.M., 2018b. Studies on influence of process parameters on simultaneous biodegradation of atrazine and nutrients in aquatic environments by a membrane photobioreactor. Environ. Res. 161, 599–608. Dizge, N., Tansel, B., 2010. External mass transfer analysis for simultaneous removal of carbohydrate and protein by immobilized activated sludge culture in a packed bed batch bioreactor. J. Hazard. Mater. 184, 671–677. Dubey, S., Nath, S., Chandra, Y., 2016. Optimization of removal of Cr by y-alumina nanoadsorbent using response surface methodology. Ecol. Eng. 97, 272–283. Geed, S.R., Kureel, M.K., Giri, B.S., Singh, R.S., Rai, B.N., 2017. Performance evaluation of Malathion biodegradation in batch and continuous packed bed bioreactor (PBBR). Bioresour. Technol. 227, 56–65. Geed, S.R., Kureel, M.K., Prasad, S., Singh, R.S., Rai, B.N., 2018. Novel study on biodegradation of Malathion and investigation of mass transfer correlation using alginate beads immobilized Bacillus sp. S4 in bioreactor. J. Environ. Chem. Eng. 6 (2), 3444–3450. Gopinath, K.P., Murugesan, S., Abraham, J., Muthukumar, K., 2009. Bacillus sp. mutant for improved biodegradation of Congo red: Random mutagenesis approach. Bioresour. Technol. 100, 6295–6300. Gusain, D., Dubey, S., Nath, S., Weng, C.H., Sharma, Y.C., 2016. Studies on optimization of removal of orange G from aqueous solutions by a novel nano adsorbent, nano zirconia. J. Ind. Eng. Chem. 33, 42–50.

Fig. 4. The spectral profile of control and biodegraded sample of Congo red in MBBR. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

1272 cm−1 due to vibration and stretching of primary aromatic amine with CeN, a peak at 1542 cm−1 and peak at1666 cm−1 due to stretching bending of azo group of N]N, a peak at 2345 cm−1 due to stretching vibration of CeC, and a peak at 3347 cm−1 due to presence of OeH group. The FTIR spectrum of CR dye treated sample shows peaks at different wave numbers; a peak at 1635 cm−1 for stretching bending of the azo group of N]N, a peak at 2347 cm−1 for stretching of CeC, and a broad peak at 3455 cm−1 due stretching vibration of OeH. In the case of CR dye treated sample, the major shift in the peaks, disappearance of peaks, and appearance of some new peaks at 532, 663, and 2067 cm−1 were observed. The FTIR result clearly showed the major difference in the spectrum of untreated and treated sample of CR dye, which supports the successful biodegradation of dye in MBBR. The GC–MS was carried out to analyze the intermediates formed during the biodegradation of CR dye. The analysis of degradation products of CR dye reveals several peaks (E-Supplementary data). These peaks indicated the formation of low molecular weight intermediate metabolites, which confirmed the degradation of the CR dye by immobilized PUF-PP Bacillus sp. MH587030.1 in MBBR. 4. Conclusions The present work elucidated the application of an MBBR bioreactor for the removal of CR dye. The optimization studies using CCD of RSM indicates that the initial CR dye concentration, pH, and carrier filling ratio adversely affected the performance of MBBR. MBBR shows excellent dye RE at optimum conditions. The dye RE was high at a low inlet loading rate in a continuous MBBR. The biodegradation kinetics was examined by a modified Stover-Kincannon model and proposed a kinetic correlation, which could be used for the prediction of effluent concentration and required volume of MBBR to scale up the process. CRediT authorship contribution statement Ravi Kumar Sonwani: Methodology, Software, Writing - original draft, Data curation. Ganesh Swain: Writing - review & editing, Investigation. Balendu Shekhar Giri: Supervision. Ram Sharan Singh: Supervision. Birendra Nath Rai: Supervision, Validation. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 8

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