Decolorization, biodegradation and detoxification of reactive red azo dye using non-adapted immobilized mixed cells

Accepted Manuscript Title: Decolorization, biodegradation and detoxification of reactive red azo dye using non-adapted immobilized mixed cells Authors: Basma B. Hameed, Zainab Z. Ismail PII: DOI: Reference:

S1369-703X(18)30163-3 https://doi.org/10.1016/j.bej.2018.05.018 BEJ 6955

To appear in:

Biochemical Engineering Journal

Received date: Revised date: Accepted date:

22-3-2018 10-5-2018 17-5-2018

Please cite this article as: Hameed BB, Ismail ZZ, Decolorization, biodegradation and detoxification of reactive red azo dye using non-adapted immobilized mixed cells, Biochemical Engineering Journal (2018), https://doi.org/10.1016/j.bej.2018.05.018 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.

Decolorization, biodegradation and detoxification of reactive red azo dye using non-adapted immobilized mixed cells Basma B. Hameed; Zainab Z. Ismail*

*

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Department of Environmental Engineering, University of Baghdad, Baghdad, Iraq

Corresponding author: Z.Z. Ismail, Emails:[email protected]; [email protected]

Highlights

Sequential anaerobic-aerobic system was used for azo dye reactive red biodegradation

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Complete decolorization of reactive red dye was accomplished during anaerobic phase

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•Non-adapted immobilized cells successfully favored the detoxification of reactive dye

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The effect of bio-carrier type on the dye decolorization efficiency was negligible

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Maximum COD removal of 96% was observed at RR2 initial concentration of 10 mg/L

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Abstract

Azo dyes are refractory and recalcitrant contaminants that pose a significant impact on the environment upon releasing to the natural resources. This study focused on the decolorization and biodegradation of water soluble azo dye reactive red (RR2) in an

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integrated batch mode sequential anaerobic-aerobic processes. Activated sludge collected from a local sewage treatment plant was used as the source of non-adapted free and immobilized mixed cells for the biodegradation of RR2. Starch as a non-conventional

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bio-carrier and the conventional alginate were alternatively used as bio-carriers to immobilize the mixed cells. The immobilized mixed cells were found to completely

decolorize 10 mg/ L of dye RR2 within 30 h under anaerobic incubation conditions. The experimental results revealed that the 96%, 93%, and 82% maximum COD removals

were observed with samples containing RR2 at initial concentration of 10, 20, and 40

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mg/L, respectively using non-adapted immobilized mixed cells. Comparable

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decolorization results were observed with both types of bio-carriers indicating that the

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effect of bio-carrier type was negligible. Additionally, UV-vis spectra demonstrated that

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the azo bond was cleaved biologically in the anaerobic phase. The acute and phytotoxicity evaluation of degraded metabolites suggests that the non-adapted

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immobilized mixed bacterial cells successfully favored the detoxification of dye RR2.

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Keywords: Reactive dye, immobilized cells, biodegradation, activated sludge,

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Decolorization

1. Introduction

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The synthetic dyes are abundantly used in several industries. The generated dyes-loaded wastewater is considered a problematic issue in many parts of the world. Nowadays, the public demand for color-free discharge has rendered decolorization of wastewater a top priority [1]. However, dyes are considered the most difficult compound to be removed

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and degraded. The presence of low concentrations of dyes in textile effluents is not only visually visible, but also results in the deterioration of both photosynthesis and the quality of aquatic life due to the carcinogenic nature of the parent dyes along with their break-

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down products [2, 3]. Azo dyes are the most widely used class of dyes with a market share of 60-70% [4]. Reactive azo dyes in particular, are most consumable type of dyes.

The main problem associated with this category is that it hydrolyzes easily which leads to a higher percentage of unfixed dye portions being washed off during the process which

necessitate treating the discarded effluent [5]. Physical and chemical treatments methods

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including adsorption, electrochemical and flocculation are frequently used for dye

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containing wastewater [6, 7], but the operational difficulties, cost-ineffectiveness and

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sludge disposal problem are of the main obstacles. Biological treatment, on the other

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hand, offers a promising opportunity for removal of organic pollutants including dyes [8, 9]. To take a further step in promoting the activity of microorganisms against the

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recalcitrant substrates, immobilization technique showed a superior activity not only in

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overcoming the toxicity of these compounds but also in simplifying the overall

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application by the reusability of the immobilized matrix, rendering the process less costly. Immobilization is one of the great tools for the development of ecologically and

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economically available biocatalyst [10, 11]. Several studies have been reported concerning the biotreatment of different types of dyes using free bacterial cells. However,

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limited investigations were reported regarding the decolorization and biodegradation of dyes using immobilized bacterial cells. Ogugbue et al. [12] studied the removal of an azo dye (Polar red B) in synthetic saline wastewater using Bacillus firmus. Cell immobilization indicated that decolorization was significantly higher in immobilized

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halotolerant cell systems than with free cells of Bacillus firmus, especially at salt concentrations higher than 4%. Kathiravan et al. [13] explored the biodegradation efficiency of alginate immobilized indigenous Aeromonas sp. MNK1 on Methyl Orange

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(MO) in a packed bed reactor. Complete MO degradation and COD removal were observed at 300 mg/l of initial dye concentration. Yogesh & Akshaya [14] studied the decolorization of Acid Maroon V using bacterial consortium EDPA containing

Enterobacter dissolvens AGYP1 and Pseudomonas aeruginosa AGYP2 immobilized in different entrapment matrices. The consortium displayed 96% removal of dye at initial

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concentration of 100 mg/L within 6 h when immobilized in agar-agar. Under optimum

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concentrations of agar-agar (3.0% w/v) and cell biomass (0.9 g% w/v), the consortium

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displayed decolorization for 18 successive batches of Acid Maroon V and also

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decolorized 14 other different textile dyes. Sharma et al. [15] investigated the capacity of Aeromonas jandaei strain SCS5 in both free and immobilized forms for methyl red (MR)

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decolorization and degradation under aerobic and anaerobic conditions. At 100 mg/L

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methyl red concentration, complete decolorization was obtained within 6 h for both

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aerobic and anaerobic conditions. Sabbir et al. [16] evaluated the bioremediation capabilities of three kinds of periphyton immobilized in bioreactors to decolorize and

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biodegrade the sulphonated azo dye, amaranth. The results demonstrated the potential application of immobilized periphyton at industrial scale for the removal of azo dyes

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from wastewater containing azo dye amaranth. Olivo-Alanis et al. [17] studied the mechanism of anaerobic bio-reduction of Congo red azo dye assisted with lawsoneimmobilized activated carbon.

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This study aimed to evaluate the potential of non-adapted immobilized mixed cells for decolorization and biodegradation of reactive red azo dye (RR2) by sequential anaerobicaerobic biotreatment processes. The potential of using starch as a non-conventional bio-

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carrier was assessed and compared to sodium alginate. To the best of our knowledge, it is the first time that starch– immobilized mixed cells with excellent decolorizing ability against azo dye reactive red has been reported.

2. Materials and methods

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2.1 Dye and microbiological media

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Azo dye reactive red (RR2) (C.I. No.18200) is widely used in textile industry. RR2

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(C19H10Cl2N6Na2O7S2) is polyaromatic di-sulphonated dichloro- triazene high molecular

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weight dye (615.324 g/mol). All other chemicals and reagents used in the study were of analytical grade. Mineral salt medium (MSM) along with various concentrations ranged

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from 0.1 to 2 g/L of yeast extract amended with predetermined RR2 concentration ranged

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from 10 to 40 mg/L was used for the development of mixed cultures. MSM was prepared

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according to the procedure outlined in Ghangrekar et al. [18] by dissolving in grams: 0.2 MgSO4.7H2O, 0.015 CaCl2, 0.001 FeCl3.6H2O, 0.02 MnSO4.H2O, 1.825 NaH2PO4, 0.35

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KH2PO4, and 0.42 NaHCO3 in liter distilled water. Glucose was used as a co-metabolite

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for azo dye decolorization.

2.2 Microorganisms Activated sludge freshly obtained from a local wastewater treatment plant in (Baghdad), was used as the source for free and immobilized mixed bacterial cells during the

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sequential anaerobic-aerobic treatment without any previous adaption to the selected reactive azo dye (RR2).

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2.3 Bio-carrier materials Two types of polymeric carriers were alternatively used in this study which were sodium

alginate as a conventional bio-carrier and the starch as a new nonconventional bio-carrier. Their abundant availability and enviromental friendliness are so tempting, however, they are known for have little stability in water. This problem can be overcomed by cross-

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linking them with polyvinyl alcohol (PVA), a commonly used, well known synthetic

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polymer.

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2.4 Immobilization protocol

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PVA is used for microorganisms entrapment by the classical freezing-thawing method

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mainly due to its effictiveness and nontoxicity towards living cells. The preparation process involves embedding the microorganisms with the PVA matrix [19]. This

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technique is superior in providing elastic, rubber like hydrogel with a limitation of the possibility of bead agglomeration due to their stickiness. This problem can be overcome

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by combining PVA with 2% sodium alginate and hardening the mixture with a solution of mixed boric acid (6%) and calcium chloride (4%) [20]. For the preparation of polyvinyl alcohol- Starch (PVA-St) matrix, a starch solution of 2% concentration was prepared and used with 13 % PVA solution. 5 ml of biomass inoculum was added to the

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PVA-Starch mixture, and shaken for few seconds without bubble formation. Then the mixture was poured into sterile micro-plates and put in the freezer. The formed beads

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were washed 3 to 4 times with distilled water [21].

2.5 Analytical methods

The UV–vis spectrum of RR2 was recorded from 200 to 1200 nm using an UV–vis

spectrophotometer (Advanced Microprocessor UV-VIS Spectrophotometer Single Beam LI-295) equipped with a quartz cell of 1.0 cm path length. The concentrations of the

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RR2samples were quantified by measuring the absorption intensity at λmax= 540 nm.

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Chemical oxygen demand (COD) was monitored using COD analyzer (Model: Lovibond,

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RD 125). Volatile suspended solids (VSS) measurements were determined according to

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the Standard Methods [22]. All measurements were performed in duplicate.

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2.6 Experimental procedure

In the present study, a sequential anaerobic-aerobic biotreatment processes were

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considered for the decolorization of RR2. For the decolorization assays, the immobilized mixed cells were incubated with RR2- loaded media in 100-mL Erlenmeyer flasks (50

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mL liquid volume). The flasks were flushed with nitrogen, firmly sealed with stoppers, manually and gently shaken at constant time intervals at room temperature (30±3°C) and

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pH range (7-7.6). At specific time intervals, 2 mL samples volumes of the supernatant were withdrawn from each Erlenmeyer flask, centrifuged at 6000 rpm for 20 min, filtered through 0.4-micron filters and measured at the maximum absorption wavelength (λmax =

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540 nm) for the remaining concentration of RR2 . The percentage decolorization was calculated according to the following formula: % Decolorization =

(1)

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Where: Ai is the absorbance of the initial concentration of RR2; Af is the absorbance of the final RR2 concentration at time t.

The effect of various parameters on the decolorization process was investigated. The

effect of yeast extract dose on decolorization process was evaluated by supplementing the

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MSM solution prepared with predetermined concentration of RR2, with different

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concentrations of yeast extract including 0.1, 0.3, 0.5, 0.7, 1, 1.5, and 2 g/L. The

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influence of RR2 initial concentration (10, 20, 40 mg/L) on decolorization was evaluated.

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In addition, the potential of RR2 decolorization was performed with different percentages of free bacterial cells, which were 3%, 5%, 7%, 10% at constant initial dye concentration

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and yeast extract dose. The effect of immobilized cells concentration on the

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decolorization process was investigated using 10% and 30% (V/V). Control experiments were prepared and manipulated in triplicate with autoclaved biomass for possible dye

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removal due to adsorption.

2.7 Reuse of beads for excessive treatment cycles

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One of the most important advantages of using immobilized bacterial cells compared to free cells is the recycling of beads without losing their activity unlike the non-reusable free cells. In this study, the immobilized mixed cells were evaluated for the potential of repetitive decolorization and subsequent aerobic biodegradation. Immobilized cells for each treatment stage were collected after use, repeatedly washed with sterilized deionized 8

water, and reused for a second cycle of decolorization and biodegradation of RR2. Similar procedure was conducted for the immobilized cells used for aerobic phase to be

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reused again for subsequent cycles.

2.8 Phytotoxicity test

Phytotoxicity tests were conducted to assess the impact of the treated supernatant on the vegetation once it is thrown to the ecosystem as well as to explore the possible reuse of the treated solution in irrigation fields. Phytotoxicity for the untreated and treated

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samples was assessed. The phytotoxicity assays using Triticum aestivum were performed

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as described by Gomare and Govindwar [23] and Zhao et al. [24]. Prior to inoculation,

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crop seeds were cleaned and surface sterilized with 3% hydrogen peroxide solution for 5

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min and then washed several times using sterilized distilled water. Four Petri-plates were prepared; each plate contained 10 healthy seeds of Triticum aestivum, and was daily

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quenched with 5-mL of aqueous solution as follows: plate (a) with tap water (as a

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control); plate (b) with 10 mg/L RR2-laden solution; plate (c) with anaerobically treated

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solution; and plate (d) with aerobically treated solution. Percentage germination, plumule and radicle length were recorded and compared. Each experiment was carried out in

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duplicate.

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3. Results and discussion 3.1 Decolorization and biodegradation analysis UV–vis scan (200-1100 nm) of RR2-loaded supernatants at different time intervals showed decolorization and decrease in dye concentration using free and immobilized

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mixed cells. The peak at 540 nm observed with the untreated RR2-loaded solution was decreased after the anaerobic biotreatement without shift in λmax up to complete decolorization using free and immobilized mixed cells (Fig. 1 A and B). The maximum

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absorbance peak at 540 nm corresponded to the existence of RR2 is related to the chromophore structure of the azo dye RR2 and a wide range band of 285-300 nm which falls within the range 280-350 nm that has been reported by Franciscon et al. [25] to be consistent with substituted benzene and naphthalene compounds.

As given by the UV scan (Fig.1), a shift of the wide band towards a sharp peak at 280 nm

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with a complete disappearance of the peak was observed in the visible region indicating

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the azo bond cleavage. The decrease in the absorbance of the band in the range of 285 -

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300 nm and the formation of a new peak at 280 nm suggested unprecedented

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transformational changes to the aromatic structure specifically to the substituents on the aromatic groups, consistent with azo bond cleavage. This observation confirmed that

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complete decolorization was achieved. However, the amines resulted from the

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biodegradation of RR2 were not degraded during the anaerobic phase, and thus a

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secondary aerobic treatment step was required to accomplish complete biodegradation. Similar observations were reported by Jonstrup et al. [26].

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Additionally, in order to investigate if there is any contribution of biosorption process along with biodegradation of RR2, an experiment was conducted with autoclaved mixed

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cells. The UV-Vis spectrum analysis revealed that the original peaks at 540 and 300 nm appeared with the RR2-loaded sample has declined by about 11% without showing new peaks (Fig.1C). This observation indicated that biosorption was initially responsible for the slight removal of RR2 which in turn facilitate and support the biodegradation process.

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Similar observation was reported by Cheng et al. [27] about immobilization of Burkholderia vietnamiensis C09V strain in PVA–alginate–kaolin gel beads as a

3.2 Effect of initial dye concentration on decolorization

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biomaterial to improve the degradation of crystal violet from aqueous solution.

The effect of initial RR2 concentration on the decolorization was investigated and it was found that the percentage of decolorization decreased with increasing the initial

concentration of RR2. By increasing the initial concentration of RR2 from 10 to 20, and

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then up to 40 mg/L the time duration for complete decolorization increased from 20 to

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25, and 40 h, respectively using free mixed cells (Fig. 2). This observation indicated that

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the decolorization rate decreased with increasing the RR2 initial concentration, but

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maintaining the overall removal efficiency intact. This may be reclined to a decrease in the growth of cultures as the initial dye concentration increased. However, extended time

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duration for RR2 decolorization was relatively observed when using immobilized mixed

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cells up to 30, 50, and 60 h at initial concentrations of RR2 of 10, 20, and 40 mg/L,

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respectively. It was noticed that after 20 h incubation, only 80% and 27% decolorization were observed when using initial dye concentrations of 20 and 40 mg/L, respectively.

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Fig. 3 presents the profiles of RR2 and COD removals over the sequential anaerobic and aerobic phases. It is well observed that RR2 was completely decolorized and biodegraded

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during the anaerobic phase regardless of its initial concentration value. These results are in disagreement with Vaigan et al. [28] when adjusting the initial concentration of reactive blue (Brill Blue KN-R) from 20 to 40 mg/L resulted in reducing their removal efficiencies of reactive dye from 57 to 31%. The trend of maintaining certain removal

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efficiency while increasing the initial concentration of the dye, has been evidenced by Cui et al. [29] when over 95% decolorization of methyl red with Klebsiella sp. strain Y3 was maintained upon increasing the initial concentration from 200 to 800 mg/L whilst the

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required decolorization time increased from 16 to 30 h, respectively. On the other hand, the anaerobic phase was prominent in reducing COD values leading up to 30 %, 60%, and 76% using PVA-SA as the bio-carrier for mixed cells at initial

concentrations of 10, 20, and 40 mg/L, in 30h, 50h, and 60h, respectively (Fig. 3). Hence, by using the bio-carrier PVA-St , the COD reduction were observed to be 53%, 58%,

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and 75% at 30h, 50h, and 60h using RR2 initial concentrations of 10, 20, and 40 mg/L,

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respectively. These observations indicated that the type of bio-carrier didn’t have notable

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effect on the efficiency of immobilized cells for RR2 and COD removals as well as the

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related time duration for mineralization. Similar results were revealed by Kapdan and Oztekin [30] who examined the validity of using sequential anaerobic-aerobic reactors

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for a synthetic wastewater with Remazol Red RR as the pollutant of interest.

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As given in Fig.3, the anaerobic stage contribution to COD removal was approximately

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50%, whilst the aerobic phase shifted the overall reduction to 80%. The much lower COD removal in the aerobic phase, compared to the anaerobic phase, could be attributed to the

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insufficient amount of readily available biodegradable substrate that remained after the anaerobic stage.

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However, increasing the initial concentration of RR2 up to 100 mg/L inhibited the free mixed cells unlike the immobilized cells which were capable of degrading RR2 at high concentration (Fig. 4). Complete decolorization indicated that RR2 did not have any inhibition effects over immobilized cells and the immobilization gels have a potential

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capacity to host the bacterial cells. The additional mass transfer resistance associated with the biocarrier slowed down the decolorization process, but meanwhile it provided the

3.3 Effect of yeast extract dose on decolorization process

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advantage of protecting the cells from the inhibition of high concentrations of the dye.

Yeast extract (YE) has a unique capacity of promoting enzyme activity compared to other frequently used sources [31]. It has been suggested in several studies that YE is the most

effective supplement for the growth of azo dye degrading bacteria, along with enhancing

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dye decolorization efficiency [32]. It has shown to be the best organic nitrogen source for

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bacterial cell growth. It is rich in growth stimulants, vitamins, and different amino acids

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making it an effective amino acids mixture, with more advantages than individual amino

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acid. The effects of yeast extract concentrations on decolorization of RR2 at 10 mg/L initial concentration by immobilized mixed bacteria are illustrated in Fig. 5. As shown in

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these plots, notable increase in decolorization efficiency was recorded upon increasing

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YE amount from 0.1 to 0.7 g/L and complete decolorization was best achieved within 30

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h by using 0.7 g/L. Further increase in YE amount reduced decolorization efficiency which might be attributed to the enzyme saturation upon 0.7 g/L of yeast extract. Similar

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observation has been reported by Imran et al. [31] who suggested that the increasing of YE above 1 g/L resulted in a slow enzyme response which shows a possible manner of

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enzyme saturation around 1g/L. This behavior might also be attributed to the fact that bacterial cultures have a tendency to consume yeast extract as a readily available nitrogen source for their growth instead of targeting the destruction of azo dye complex structure.

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3.4 Effect of inoculum size The effect of inoculum size on RR2 decolorization was conducted by inoculating the MSM with 3%, 5%, 7%, and 10% (v/v) mixed cells. As given in Fig. 6, inoculum sizes of

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3% and 5% showed 84% decolorization efficiency after 30 h of anaerobic incubation. Upon increasing the inoculum size to 7%, the removal efficiency has escalated to reach 100% after only 20 h of anaerobic decolorization suggesting the effective impact of the

availability of more active sites to accomplish the degradation process. Inoculum size of

10% seems to make no notable difference in decolorization duration compared to that of

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7%. Similar trend of increasing decolorization with increasing the inoculum size pattern

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has been observed by Sani and Banerjee [33]. However, beyond 10% (v/v) inoculum size,

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the rate of increasing decolorization was not very significant which suggests no

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proportionate increase in decolorization percentage with increasing bacteria inoculum

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size of Kurthia sp.

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3.5 Recycling of immobilized mixed cells

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Recycling of beads was examined as a promising strategy with a greater reusability for reactive dyes removal. As given in Fig. 7, the time duration required for complete

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decolorization during the anaerobic treatment of RR2 was about 30 h with a relatively notable reduction in COD removal rate during the second cycle. This might be attributed

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to the partial blockage of bead pores caused by substrate biodegradation leading to a limited access of dye particles to the active sites. Daassi et al. [34] claimed that the slightly diminished activity, specifically in alginate beads, could be explained in terms of bead washing which presumably causes enzyme leakage.

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As opposed to the anaerobic phase, the aerobic phase of the second cycle offered a higher COD removal % that ultimately resulted in complete mineralization, not to mention the reduction in the required time course. Both PVA-SA and PVA-St beads exhibited a

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behavior in the second cycle that is peculiarly better than that of the first cycle. This trend mirrored the final COD removal percentages. The highest COD removal percentages for both PVA-SA and PVA-St were 85% after 67 h, whereas the second cycle resulted in

complete COD removal within only 47 h. The reason could be credited to the cultures’

induced ability to eject aerobic enzymes which are responsible for aromatic ring cleavage

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under aerobic conditions. Yaşar et al. [35] have noted that catechol 2, 3-dioxygenase

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(C23DO), the aerobic enzyme used as an indicator for aerobic biodegradation of aromatic

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amines, has experienced an increase as the length of the aerobic period increased.

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Accordingly, the longer the aerobic period, the greater was the induction. The bottom line is that subsequent treatment cycles exhibit an advantage over the first cycle by allowing

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enzyme induction.

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the cultures to maintain in the same preferable environment leading to a higher aerobic

3.6 Phytotoxicity test

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The results of toxicity test are presented in Table 1. The germination of triticum aestivum was 30% using RR2-loaded solution which was relatively low compared to100%

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accompleshid with tap water as the control solution. The results revealed that by accomplishing the anaerobic treatment stage and using the resulted solution for plant watering, the germination rate increased to 60 %. An outstanding result was observed by using the aerobically-treated solution with a germination rate up to 100%. The plumule

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and ridicule lengths were 0.5 cm and 0.2 cm with the RR2-loaded solution, where the anaerobically treated solution showed more promising results with 1.32 cm and 0.6 cm, respectively. Against all odds, the aerobically treated solution stood out over tap water,

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showing superior results with 2.4 cm and 1.8 cm, compared to 1.1 cm and 0.5 cm in terms of plumule and radicle lengths, respectively. Those results might be due to the minerals content of the MSM which works in the favor of plant growth.

4. Conclusion

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The biodegradation of azo dye reactive red (RR2) was performed by using non-adapted

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mixed bacterial cell culture cultivated from activated sludge produced from local

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wastewater treatment plant. The experimental decolorization and degradation capacity for

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the RR2 was found 100% using free and immobilized mixed cells at 20 and 30 h, respectively under anaerobic conditions. The experimental results revealed that the 96%,

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93%, and 82% maximum COD removals at RR2 initial concentration of 10, 20, and 40

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mg/L, respectively using non-adapted immobilized mixed cells. In the present

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investigation, mixed cells have been alternatively immobilized in starch and alginate as non-conventional and conventional bio-carriers, respectively without losing their viability

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or ability to decolorize the RR2 even upon recycling them for a second treatment cycle. Also, using commercial type of RR2 supports choosing the suggested approach as a

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reliable method to be directly adopted at industrial scale.

Acknowledgment

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The authors would like to acknowledge the support of the State Company for Textile Industry, Ministry of Industry in Iraq for their technical support.

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azo dye degrader. J. App. Microbiol. 106 (2009) 993-1004.

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characterization, and enzyme analysis. Environ. Sci. Pollut. Res. 21(2014) 6136–6145. [25] E. Franciscon, M.J. Grossman, J.A.R. Paschoal, F.G.R. Reyes, L.R. Durrant,

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Tables

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Figures

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Table 1 Phytotoxicity test results for watering Triticum aestivum with tab water, RR2loaded solution, anaerobically-treated solution, and aerobically-treated solution

A

Fig. 1 UV-vis spectra; (A) Free mixed cells, (B) immobilized mixed cells, (C) blank beads (without cells) Fig. 2 Effect of RR2 initial concentration on decolorization; immobilized cells, alginate-immobilized cells

free cells,

starch-

Fig. 3 Effect of RR2 initial concentration on the remaining concentration of RR2 and % COD removal

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Fig. 4 Comparison between free cells and immobilized cells for RR2 biodegradation at 100 mg/L initial concentration Fig. 5 Effect of YE dose (g/l) on the RR2 decolorization at 10 mg/L initial concentration

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Fig. 6 Effect of inoculum size (v/v) on RR2 decolorization at 10 mg/L initial concentration

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D

M

A

N

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Fig. 7 The effect of recycling on COD removal

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23

D

TE

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N

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M

A

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TE

D

M

A

N

U

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Fig. 1 UV-vis spectra; (A) Free mixed cells, (B) immobilized mixed cells, (C) blank beads (without cells)

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starch-

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M

A

N

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Fig. 2 Effect of RR2 initial concentration on decolorization; free cells, immobilized cells, alginate-immobilized cells

25

3

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TE

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M

A

N

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Fig.

A

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Effect of RR2 initial concentration on the remaining concentration of RR2 and % COD removal

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Fig. 4 Comparison between free cells and immobilized cells for RR2

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A

N

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biodegradation at 100 mg/L initial concentration

27

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D

M

A

N

U

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Fig. 5 Effect of YE dose (g/l) on the RR2 decolorization at 10 mg/L initial concentration

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Fig. 6 Effect of inoculum size (v/v) on RR2 decolorization at 10 mg/L initial concentration

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Fig.7 The effect of recycling on COD removal

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Table 1 Average phytotoxicity test results for watering Triticum aestivum with tap water, RR2-loaded solution, anaerobically-treated solution, and aerobically-treated solution Parameters

Tap water

RR2-loaded

Anaerobically-

Aerobically-

solution

treated solution

treated solution

60

100

100

30

Plumule (cm)

1.1

0.5

1.32

2.4

Radicle (cm)

0.5

0.2

0.6

1.8

A

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TE

D

M

A

N

U

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Germination (%)

30