Ecotoxicology and Environmental Safety 83 (2012) 108–114

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Ecotoxicology and Environmental Safety

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Biodegradation of crystal violet using Burkholderia vietnamiensis C09Vimmobilized on PVA–sodium alginate–kaolin gel beads

Ying Cheng a, HongYan Lin a, Zuliang Chen a,b,c,n, Mallavarapu Megharaj b,c, Ravi Naidu b,c

a School of Environmental Science and Engineering, Fujian Normal University, Fuzhou 350007, Chinab Centre for Environmental Risk Assessment and Remediation, University of South Australia, Mawson Lakes, SA 5095, Australiac Cooperative Research Centre for Contamination Assessment and Remediation of Environments, Mawson Lakes, SA 5095, Australia

a r t i c l e i n f o

Article history:

Received 20 January 2012

Received in revised form

19 June 2012

Accepted 19 June 2012Available online 11 July 2012

Keywords:

Biodegradation

Crystal violet

Burkholderia vietnamiensis C09V

Immobilisation

Polyvinyl alcohol (PVA).

13/$ - see front matter & 2012 Elsevier Inc. A

x.doi.org/10.1016/j.ecoenv.2012.06.017

esponding author at: Centre for Environm

ation, University of South Australia, Mawso

1 08 83023057.

ail addresses: Zuliang.chen@unisa.edu.au, zlch

a b s t r a c t

The strain, Burkholderia vietnamiensis C09V was immobilized on PVA–alginate–kaolin gel beads as a

biomaterial to improve the degradation of crystal violet from aqueous solution. The results show that

98.6% (30 mg L�1) crystal violet was removed from aqueous solution using immobilized cells on PVA–

alginate–kaolin gel beads, while 94.0% crystal violet was removed by free cells after degradation at the

pH 5 and 30 1C for 30 h. Kinetics studies show that the pseudo-second-order kinetics well described the

adsorption of crystal violet on the PVA–alginate–kaolin beads. Biodegradation of crystal violet on

immobilized cells was fitted well by first-order reaction kinetics, indicating that CV was adsorbed onto

kaolin and followed their degradation by immobilized cells onto the the PVA–alginate–kaolin beads.

Characterization with SEM shows that cells attached well to the surface of PVA–alginate–kaolin beads,

leading to improved crystal violet transfer from aqueous solution to immobilized cells. In addition,

UV–vis show that the absorption peak at 588 nm was reduced by the degraded N-bond linkages, as well

as the formation of degrading products were observed by Fourier transform infrared (FTIR). These

results suggest that crystal violet was biodegraded to N,N-dimethylaminophenol and Michler’s Ketone

prior to these intermediates being further degraded.

& 2012 Elsevier Inc. All rights reserved.

1. Introduction

Crystal violet as a triphenylmethane dye is often used for textiles,printing, papermaking, leather, food, and cosmetics (Ayed et al.,2009). Since it is carcinogenic and persists in a variety of environ-ments, the removal of crystal violet from wastewater has become amajor environmental concern in recent years (Chen et al., 2008a,2008b; Daneshvar et al., 2007). A wide range of methods has beendeveloped to remove synthetic dyes from wastewaters to alleviatedamage to the environment. The technologies involve adsorption,photocatalysis oxidation and biodegradation (Forgacs et al., 2004).An alternative has been suggested in that degradation of dyes usingbiological methods should be more economical, environmentallyfriendly and complete despite this representing a slower rate ofbiodegradation (Venkata et al., 2007).

A number of microorganisms have been isolated and character-ized as being able to degrade crystal violet (Chen et al., 2008a,2008b; Daneshvar et al., 2007). However, free cells used in biode-gradation are limited in terms of rate, operational stability, cellwashout and transfer of substrate into the cell (Ha et al., 2009). To

ll rights reserved.

ental Risk Assessment and

n Lakes, SA 5095, Australia.

en@fjnu.edu.cn (Z. Chen).

address these issues, immobilized cells on various matrices couldrepresent a potentially and effective technique in biodegradationbecause immobilized cells tend to have a higher level of activity.They are also more resilient to environmental perturbations such aspH, or exposure to toxic chemical concentrations than suspensioncells, and more favourable for oxygen supply and mass transfer(Rodrı́guez-Couto, 2009; Steffan et al., 2005).

Cell entrapment is one of the most used techniques for cellimmobilization, where the cells are trapped in natural gels suchas alginate, and carrageenan synthetic gels such as polyvinylalcohol and polyacrylamide (Rodrı́guez-Couto, 2009). Of thesemethods, entrapment in alginate beads has become a preferredtechnique for the degradation of dyes (Dominguez et al., 2005;Say et al., 2001; Steffan et al., 2005). These drawbacks have driventhe search for other types of support materials such as PVA andkaolin. PVA, for example, is a preferable immobilization matrixbecause its porous structure allows the substrate and oxygen todiffuse into the internal pores and makes biodegradation possible(Al-Zuhair and El-Naas, 2011). Kaolin, which is a porous materialand efficient adsorbent, has great potential in removing cationicdyes from wastewaters because it has chemical and mechanicalstability, high adsorption capacity and is not toxic to cells (Nandiet al., 2009a). On the other hand, integration of porous materialssuch as PVA and adsorbents such as kaolin into beads can alsofacilitate the transport of the crystal violet toward both the

Y. Cheng et al. / Ecotoxicology and Environmental Safety 83 (2012) 108–114 109

surface and the interior. It could provide certain advantages forgrowing cells in terms of reusability, freedom from toxicity,mechanical strength and open spaces within the matrix.

In this study, a new strain was isolated from activated sludgeand identified as Burkholderia vietnamiensis C09V (B. vietnamiensis

C09V) and was used to biodegrade crystal violet from aqueoussolution. However, since the freely suspended cells used inbiodegradation were limited due to their low efficiency and pooroperational stability (Ha et al., 2009), the immobilized B.vietna-

miensis C09V cells were developed as an alternative for thebiodegradation of crystal violet. Since the addition of kaolin tobead increases in adsorption of crystal violet to the bead, as wellas improves beads to maintain their physiological stability andmechanical strength. The mechanism in the degradation of CV byimmobilized cells bead may be include the adsoption of CV on thebead and then degradation by the cell immobilized on bead.To our knowledge, there are no reported applications of B.vietna-miensis C09V immobilized on PVA–alginate–kaolin beads for thedegradation of crystal violet. Therefore, the objectives of thisstudy include: (1) comparing the degradation of crystal violetusing both free cells and immobilized cells, as well as thecondition affecting the degradation of crystal violet using immo-bilized cells; (2) the elucidation of kinetics for both adsorption ofcrystal violet on the PVA–alginate–kaolin beads and the biode-gradation of cells immobilized on PVA–alginate–kaolin beads;and (3) the characterization of biodegradation using the SEM, UVand FTIR to understand the pathway of the biodegradation.

2. Materials and methods

2.1. Chemicals and culture media

All the chemicals used in the experiments were analytical reagent grade.

Crystal violet (CV, purityZ96%) was purchased from Sigma-Aldrich and used

without further purification. PVA with an average degree of polymerization of

2400–2500 were purchased from Shanghai Chemical Factory in China. Kaolin with

135 mm particle size was obtained from Kaolin Company which is based in

Longyan, Fujian Province, China.

The phosphate-buffered basal medium (BM) (Saltikov et al., 2003) was

composed of (gL�1 of distilled water unless otherwise indicated): K2HPO4, 1.8;

NaH2PO4 �12H2O, 3.0; MgCl, 0.1; KNO3, 1.0 and glucose 3.0. Solid BM plate

contains (g L�1 BM): agar, 20. Luria–Bertani medium (LB medium) which was

composed of (g L�1): NaCl, 5; peptone, 10; yeast extract was used for aerobic

cultivation at 30 1C. Solid BM plate contains (g L�1 BM): agar, 20. Flasks containing

the medium were sterilized by autoclaving at 121 1C for 20 min.

2.2. Isolation and identification

B.vietnamiensis C09V, a facultative bacterium, was originally isolated from

wastewater sludge collected from the Textile Printing & Dyeing Mill based at Fuan,

Fujian in China. The isolated strain was identified using Gram staining, morphol-

ogy characters and biochemical tests. Further identification was done employing

16S rRNA gene sequencing. A fragment of 16S rRNA was amplified using a Gene

Amp PCR System (PE, USA). Sequencing on both strands of PCR-amplified

fragments was carried out using the dideoxy chain termination method in an

ABI 3730 automated sequencer (Invitrogen, Shanghai, China). Using the Basic

Local Alignment Search Tool (BLAST) program (Nievas et al., 2006), 16S rRNA

homology searches of the NCBI database were carried out, the aim being to find

similar sequences in the 16S rRNA database. The result indicated that the 16S

rDNA sequences of the strain had up to 100% similarity with a culture of

Burkholderia vietnamiensis. The 16S rRNA gene sequence of B.vietnamiensis C09V

was deposited at GenBank under Accession No. JF922108. Batch experiments

showed that more than 98.86% was degraded within 42 h at pH 5 and 30 1C.

During the culture period, bacterial growth was monitored by spectrophotometry

at 600 nm (Chen et al., 2009).

2.3. Immobilization

Polyvinyl alcohol (PVA) and sodium alginate were dissolved in distilled water

at 80 1C, and after that kaolin (about 135 mm in diameter) was dissolved. The

formed mixture was allowed to cool to room temperature before adding 10 mL of

the previously prepared bacterial suspension.The OD600 of the bacterial suspen-

sion was 0.7. It was then well stirred for 10–15 min to ensure the homogeneity of

the whole solution. The mixture was dropped into boric acid (3% w/v) and CaCl2

(4% w/v) solution and kept for 20 h to form gel beads (about 3 mm in diameter).

The formed particles were cultured in the heterotrophic medium for 48 h and

washed in sterilized water three times, then finally stored in distilled water at 4 1C

for further use (Chen et al., 2009).

2.4. CV degradation experiment

The degradation of crystal violet was conducted as follows: 2.5 g beads were

aseptically added into 50 mL flask containing 30 mL sterilized medium at optimal

conditions (kaolin 2.5%, PVA 10%, sodium alginate 0.3%, microorganisms 10%,

CaCl2 4%, cross-linking time 20 h, cultivation time 36 h). Free cells were also

inoculated so that they could be compared with immobilized cells. Samples were

withdrawn periodically to analyze crystal violet concentration. All experiments

were performed in triplicate and were performed under forced aeration in a

gyratory incubator at 150 rpm (30 1C ) in the dark.

2.5. Analytical method and characterization

Crystal violet concentration was measured using a UV-Spectrophotometer

(722 N, Shanghai, China) at 588 nm. The removal efficiency of crystal violet was

calculated using the following equation (Wang et al., 2007):

%Decolorization¼A0�Ae

A0� 100 ð1Þ

where A0¼the initial absorbance of the medium; and Ae¼the absorbance of

decolorized medium.

Fourier transform infrared spectra (FTIR) concerning the ‘before’ and ‘after’

phases of biodegradation of crystal violet were obtained by using an infrared

spectroscope (FTIR Nicolet 5700, Thermo Corp., U.S.A). Samples for FTIR measure-

ment were prepared by mixing 1% (w/w) dry biomass with 100 mg of KBr powder

and pressed into a sheer slice. An average of 16 scans was collected for each

measurement with a resolution of 2 cm�1.

SEM images were taken at 10 kV using a Scanning Electron Microscope

(HitachiS-570, Japan). The scanning electron micrographs were taken on a JSM-

6100, JEOL (Japan) scanning electron microscope. The particles were then coated

with gold powder and attached onto the microscope supports with silver glue

(Harris et al., 2001; Zhang et al., 2007).

Aliquots of cell cultures (50 ml) collected after decolorization of crystal violet

(100 mg L�1) then centrifuged at 10,000 g for 10 min, and the supernatant

collected was extracted with 100 ml of ethylacetate. The extracts were dried

and evaporated with nitrogen gas until it dry, then acetonitrile were added prior

to LC/MS analyses. Metabolites were identified using liquid chromatography-mass

spectrometry(HPLC/Q-TOF-MS, Bruker, Germany). A C18 analytical column

(3.0�50 mm, 2.2 mm particle size) was used doe the separation at 30 1C with a

injection volume was 10.0 uL. The mobile phase consisted of a mixture of

(A) methanol and (B) 5 mmol L�1 ammonium acetate in MilliQ water with a flow

rate was 0.3 mL min�1. A linear gradient was applied starting at 35% of (A), to 45%

of (A) in 2 min, up to 90% in the next 7 min and at 90% for 2 min, then to 35%

in 0.1 min and stay at 35% for 2 min. The mass spectrometer was operated

in positive ESI mode. The source conditions were as follows: End Plate Offset:

�500 V, 91 nA; Capillary:4500 V, 4 nA; Nebulizer:0.6B bar; DryGas:6.0 L min�1

and Dry temperature:180 1C.

3. Results and discussion

3.1. Biodegradation of crystal violet by free cells and

immobilized cells

The degradation of crystal violet using free cells, immobilizedcells and control beads (sterile beads without immobilizedB.vietnamiensis C09V) was compared. As shown in Fig. 1, thehighest degradation efficiency of crystal violet (97.2%) wasachieved by the immobilized cells, while 93.1% of crystal violetwas degraded by free cells after 30 h degrading. This indicatesthat the suspended cells effectively degraded the crystal violet,where more than 96.9% CV was degraded within 42 h at pH 5 and30 1C after a short adaptation period. Some microorganisms canbe used to biodegrade crystal violet, including Shewanella deco-

lorationis NTOU1 (Chen et al., 2010) and Staphylococcus epidermidis

(Ayed et al., 2010). However, the new strain B.vietnamiensis C09Vused to biodegrade crystal violet has been not reported. This

Fig. 1. Decolorization of crystal violet by free cells, immobilized cells and sterile

immobilized control (The initial concentration: 30 mg L�1; agitation speed:

150 rpm; pH: 5; temperature: 30 1C ).

Table 1The kinetic constants of crystal violet adsorption by sterile PVA–alginate–

kaolin gels (Agitation speed: 150 rpm; pH: 5; temperature: 30 1C).

Initial concentration(mg/L)

qe

(mg/g)K(g/mg �min)

R2

20 9.21 0.0101 0.992

60 29.50 0.0031 0.994

100 53.19 0.0009 0.990

150 75.76 0.0008 0.998

Y. Cheng et al. / Ecotoxicology and Environmental Safety 83 (2012) 108–114110

study therefore presents the first demonstration where crystalviolet can be effectively biodegraded using the B.vietnamiensis

C09V strain.In comparison to immobilized cells, 76.9% crystal violet was

removed by sterile beads and the adsorption of crystal violetonto the sterile beads reached equilibrium within 6 h. This out-come echoes an earlier study where crystal violet adsorbed ontokaolin (Nandi et al., 2009b), and indicated that kaolin was aneffective adsorbent for removing crystal violet from aqueousmedium. This is attributed to crystal violet adsorbing onto kaolinusing electrostatic interaction between crystal violet and thesurfaces of kaolin with a constant negative charge(Nandi et al.,2009b). Furthermore, calculated external mass transfer coefficientshowed that crystal violet dye adsorbed more quickly onto thekaolin.

The degradation rate of crystal violet using immobilized cellswas higher than that of free cells in the first 20 h, thus indicatingthat immobilized cells on the PVA–alginate–kaolin beads signifi-cantly improved the degradation of crystal violet. This findingagrees with other cells being immobilized on alginate for thedegradation of dyes (Chen and Lin, 2007; Steffan et al., 2005).However, in this study, the PVA and kaolin in beads had a porousstructure, allowing the substrate and oxygen to diffuse into theinternal pores and retaining a high mechanical strength (Chenand Lin, 2007). Consequently, the degradation of crystal violetusing immobilized cells was significantly improved compared tothat of the free cells. This is attributed to the fact that immobi-lized cells in the PVA and kaolin in beads could increase thestability of the microbial system and maintain a high level ofcrystal violet degradation longer than the free cells (Tao et al.,2009). It was also evident that immobilizing material such asPVA–alginate–kaolin can alter cell physiology and increase cellmembrane permeability. These materials and the external cells ofmicrocolonies on the surface of gel beads act as a barrier thatprotects internal cells (Ha et al., 2009).

In summary, the immobilized cells are superior to both kaolinand free cells in the degradation of crystal violet. During the first6 h, the freely suspended cells degraded only 15.6% crystal violet,while 80.9% crystal violet was degraded by immobilized cells, ofwhich there are slight more than the control beads (76.9%). It canbe concluded that adsorption of crystal violet onto the

immobilized cells plays an important role during the first 6 hsince the kaolin can be used for the adsorption of crystal violet.The rate of removal of crystal violet using immobilized cells wasmuch higher than that of free cells, which is attributable to thecrystal violet being adsorbed onto kaolin; following their degra-dation by immobilized cells (Kulkarni and Chaudhari, 2007).

3.2. Kinetics of CV degradation

In the previous section, the removal of crystal violet usingimmobilized cells resulted from crystal violet firstly adsorbingonto kaolin and second the degradation by the immobilized cells.Hence, adsorption and biodegradation kinetics are investigated inthis section to further confirm the mechanism.

3.2.1. Adsorption kinetics

To understand the adsorption of crystal violet on the surface ofPVA–sodium alginate–kaolin, the pseudo-second order model isused to find the model that best fits the experimental data so thatthe controlling mechanism for the adsorption process can beexamined. This controlling mechanism is based on: first, thesorption processes for crystal violet on the kaolin; and second,the adsorption capacity of the adsorbent (Nandi et al., 2009b).In this study, the pseudo-second order kinetic model fitted best tothe experimental data. The pseudo-second order kinetic model isrepresented as (Nandi et al., 2009b):

dq

dt¼ Kðqe�qÞ2 ð2Þ

Integration of eq. (2) gives:

t

q¼

t

qe

þ1

Kq2e

ð3Þ

where qe is the adsorbing capacity of beads at equilibrium; q isthe adsorbing capacity of beads at time t (min); t is time (min);and K is the second order rate constant. When t/q is differentiatedby t, and 1/qe is obtained from Y-intercept, then the value of K canbe calculated.

The calculated values of K, qe and their corresponding regres-sion coefficient (R2) values are presented in Table 1. The value ofregression coefficients (R2) is more than 0.99 and the calculated qe

values ranged from 9.21 to 75. 76 mg g�1 when the initialconcentration of crystal violet increased from 20 to 150 mg g�1.This confirms that the sorption kinetics of crystal violet follows apseudo-second order process, and agrees with a previous study’sanalysis of crystal violet being adsorbed onto the kaolin (Nandiet al., 2009a). It has been observed that absorption capacity andinitial adsorption rate increased significantly when the initialcrystal violet concentration also increased, while the rateconstant fell from 0.0101 to 0.0008 g mg�1 min�1. This outcomecan be explained by the fact that there would be increasedcompetition for the active adsorption sites and the adsorptionprocess would increasingly slow down as crystal violet concen-tration increased (Nandi et al., 2009a). Due to the increasing

Y. Cheng et al. / Ecotoxicology and Environmental Safety 83 (2012) 108–114 111

driving force of the concentration gradient with a simultaneousincrease in initial dye concentration, the value of qe rose from 9.21to 75.76 mg g�1. This result is consistent with previous findingsin adsorption studies (Ahmad and Kumar, 2010).

3.2.2. Biodegradation kinetics

A number of models have been applied to describe thebiodegradation of organic pollutants. Of these models the first-order kinetic model is often used to describe biodegradation ofdyes (Chen and Lin, 2007). In this study, biodegradation of crystalviolet by free cells and immobilized cells were fitted to the first-order kinetic equation, which has the following form:

�lnC

C0¼ kt ð4Þ

where c is the concentration of crystal violet (mg L�1) at time t (h);k is the biodegradation rate constant; and A is a constant.

The half-life for biodegradation of crystal violet can beexpressed as follows:

t1=2 ¼ln 2

kð5Þ

The biodegradation of crystal violet by free and immobilizedcells under various temperatures was investigated, where thetime taken to degrade crystal violet using immobilized cells wasshort compared to that free cells. The data from the kineticsanalysis for the biodegradation of crystal violet are listedin Table 2. It can be concluded that the kinetics of degradingcrystal violet by both immobilized cells and free cells werewell expressed by the first-order model since a high-degreelinear relationship (R240.99) was found for the � log [C/C0] andtime. When the temperature rose from 25 to 35 1C, the degrada-tion rates of free and immobilized cells increased from 0.020to 0.044 mg L�1 h�1 and from 0.041 to 0.060 mg L�1 h�1,respectively. However, the values of the biodegradation rateconstant k by free cells significantly increased compared tothat of immobilized cells. This indicated that immobilized cellswithin PVA–alginate–kaolin beads could provide a better thermalstability during storage and operation than free cells (Wu et al.,2009).

As listed in Table 2, a smaller value of half-life t1/2 wasobtained using immobilized cells instead of free cells at a testedtemperature, indicating that immobilized cells were less sensitiveto changes in environmental conditions such as temperature. Insummary, comparison of the biodegradation rate constant k andhalf-life t1/2 of the immobilized cells and free cells suggests thatthe biodegradation of crystal violet by the former was much fasterthan the latter and had a shorter half-life. This finding isconsistent with other studies on beads using immobilized cellsfor the biodegradation process (Chen and Lin, 2007).

Table 2The kinetic equations of crystal violet degradation by free and immobilized cells

(The initial concentration: 60 mg/L; agitation speed: 150 rpm; pH: 5.).

Temperature (1C) K (h�1) t1/2(h) R2

Free cells 25 0.020 34.7 0.991

30 0.038 18.2 0.996

35 0.044 15.8 0.992

Immobilized cells 25 0.041 16.9 0.991

30 0.042 16.5 0.997

35 0.060 11.6 0.994

3.3. Factors affecting the biodegradation of crystal violet

3.3.1. Effect of temperature

To confirm the finding that immobilized cells and free cells cantolerate temperature changes, crystal violet degradation experi-ments were performed at temperatures ranging from 25 to 35 1Cat pH 5.0 with a crystal concentration of 60 mg L�1. The datashows that the degradation efficiency of free and immobilizedcells increased when the temperature rose from 25 to 35 1C. Theoptimal temperature was the same for cases (35 1C), where thedegradation efficiency was 75.8% and 95.3% for free and immo-bilized cells, respectively. In the 25–35 1C range, the immobilizedcells showed a higher value of degradation constants than that offree cells. However, the degradation efficiency using free cellssignificantly improved when the temperature changed. Thisverified that temperature exerted less influence on immobilizedcells than free cells since the immobilization of microorganismscan protect them from harsh environmental conditions (Chenet al., 2007; Wu et al., 2009). The results from the immobilizedcells demonstrate their better thermal stability during operationsthan the free cell system, indicating that PVA–alginate–kaolinbeads have a protective effect in their maintenance of cell activity(Chen and Lin, 2007).

3.3.2. Effect of initial dye concentration

Effect of initial crystal violet concentrations on biodegradationwas also examined to assess their capacity to tolerate differentconcentrations of crystal violet. The data shows that the immo-bilized cells displayed better removing efficiencies. The adapta-tion period for free cells increased with higher crystal violetconcentrations, and degradation efficiency decreased from 98.7%to 46.9% while the value of OD600 of strain C09V descended from1.274 to 0.495. In comparison the degradation efficiency of crystalviolet using PVA–alginate–kaolin beads changed from 99.7% to95.2% with the initial crystal violet concentrations increased from20 to 150 mg L�1. However, crystal violet had much more inhibi-tion in mouse cells than in strains. Date show that crystal violetwere cytotoxic to the mouse cell, and cell number of mouse cellclone L-929 in 4-well plates after incubation with crystal violetfor 48 h varied from 7.5þ0.30�105 to 6.67þ5.80�103 cells L�1

while the concentration of crystal violet rose from 20 to100 mg L�1 (Chen et al., 2008a, 2008b) The toxicity of crystalviolet at high concentrations could inhibit the growth of free cells.and the carrier material of the immobilized cells could act as aprotective barrier against the toxicity of crystal violet.

This indicates that the immobilized cells could sustain highertoxicity caused by larger amounts of crystal violet since theimmobilized cells had more potential to degrade crystal violet.This is due to the fact that immobilized cells affect the physiolo-gical responses of cells to xenobiotic metabolism, includingenhanced induction of the degrading enzymes, increased specificcell growth and cell yield during the crystal violet biodegradationprocess (El-Naas et al., 2009). Furthermore, as shown inTables 1 and 2, the biodegradation kinetic rate constants usingeither free cells and immobilized cells is much higher than thoseusing sterile beads, indicating that biodegradation rather thanphysical adsorption prevailed for immobilized beads. This wasparticularly the case concerning high initial crystal violetconcentrations.

3.3.3. Effect of reuse

Repeated use is an important advantage of immobilized cellssince it can reduce wastage of cells, save time and reducecultivation costs (Wu et al., 2009). Reusing immobilized cells todegrade crystal violet was done by adding the immobilized cell

Y. Cheng et al. / Ecotoxicology and Environmental Safety 83 (2012) 108–114112

beads into a 30 mL medium containing 60 mg/L crystal violet.After 24 h biodegradation, the previous medium was decantedand beads were washed with water three times before beingtransferred into a 30 mL fresh medium. As shown in Fig. 2, thedegradation rate increased from 95.4% to 99.1% in the first threecycles, then a high decolorization rate of 99.4% occurred at the 6thcycle. Degradation efficiency stayed at a level higher than 91.1%during the 10th repeated-batch experiment. The remarkableincrease in the degradation rate in the first three cycles indicatedthat cells became better adapted to the reaction conditions asbatches were repeated (Ha et al., 2009). Following this thedegradation efficiency was maintained stably and the immobi-lized cells could be reused for a maximum 10 cycles, during whichthe beads maintained their physiological stability and mechanicalstrength. When the tenth treatment was completed, beads startedweakening and cell leakage occurred. This has been documentedin other studies (Chen et al., 2008a, 2008b; Wu et al., 2009). Theimmobilized cells’ high degradation efficiency and stability duringthe reuse test suggest that the immobilization technique has thepotential for practical application.

3.4. Characterization of the biodegradation

3.4.1. SEM images of beads

Fig. 3 depicts the images of the free cells and the surface ofPVA–alginate–kaolin beads (before and after cell immobilization)using SEM. Based on the micrographs, the size of the short

Fig. 2. Repeated crystal violet degradation by reuse of the immobilized cells.

(The initial concentration: 60 mg L�1; agitation speed: 150 rpm; pH: 5; tempera-

ture: 30 1C).

Fig. 3. SEM photographs of cells and beads. (a) Free cells 5000� ; (b) Sterile cont

of CV 5000� .

rod-shaped cells was about 1 mm�0.5 mm in both Fig. 3(a) and(c), which was a typical size for bacteria. As shown in Fig. 3(c), thecells grew well on the surface of beads which indicated thatB.vietnamiensis C09V could be successfully entrapped in PVA–alginate–kaolin beads and biomass was evenly distributed withinthe bead. This result is consistent with previous findings on theuse of Ca–alginate beads for removing uranium from aqueoussolution (Akhtar et al., 2009).

Fig. 3(b) reveals that the sterile gel contains an amorphousregion, porous structures and fine spherical particles of a few mmin diameter. A number of macropores that can provide enoughspace to deliver oxygen, substrates and metabolites appear inimmobilized beads, since kaolin particles, PVA filaments and thecells are closely interlinked (Fig. 3(c)). It was consequentlyassumed that the structure of the immobilized gels could accel-erate the mass transfer of substrate from the environment to thecentral reaction site, which resulted in speeding the degradationof crystal violet (Wang et al., 2009; Wang et al., 2007).

3.4.2. UV –visible scanning

Fig. 4 illustrates the UV–vis spectra of crystal violet degraded byimmobilized cells on the PVA–alginate–kaolin beads at 0, 24, 30 and36 h, where UV–vis wavelength scanned from 200 to 800 nm andthe solution contained an initial concentration of 30 mg/L.The spectra of crystal violet exhibit large peaks with a maximumat 588 and 300 nm, corresponding to the –C¼C– bond and aromatic

rol after adsorption of CV 5000� (c) Immobilized beads after biodegradation

Fig. 4. UV–vis spectra of crystal violet biodegraded by immobilized B. vietnamiensis

C09V.

Y. Cheng et al. / Ecotoxicology and Environmental Safety 83 (2012) 108–114 113

rings (Noubactep, 2009). The peaks at 588 and 300 nm significantlydeclined after 24 h biodegradation while a new peak at 360 nm wasobserved after 30 h biodegradation, resulting from the formation ofthe metabolites of crystal violet (Asad et al., 2007). These indicatethat the removal of crystal violet by the immobilized cells on thePVA–alginate–kaolin includes both adsorption and biodegradation,where peaks at 588 and 300 nm decrease approximately in propor-tion to each other in the adsorption of crystal violet onto theimmobilized cells. In contrast the major peaks disappeared com-pletely and a new peak appeared which resulted from the biode-gradation of crystal violet.

3.4.3. FTIR spectra changes

Before and after biodegradation of the crystal violet by theimmobilized cells on the PVA–alginate–kaolin beads, scans were

Fig. 5. FTIR spectra of crystal violet biodegraded by immobilized B. vietnamiensis C09V.

Fig. 6. LC–MS spectra obtained from samples before and after biodegradation for 24

references to color in this figure legend, the reader is referred to the web version of th

done in the range of 4000–400 cm�1 wave numbers using a FTIR.Spectra are shown in Fig. 5 and they indicate differences inchanges in bands before and after the biodegradation process.Before biodegradation (Fig. 5(a)), the bands at 3440 and2918 cm�1 corresponded to N–H stretching vibration, the bandat 1590 cm�1 corresponded to C¼C stretching vibrations, theband at 1375 cm�1 corresponded to CH3 asymmetric bend, andthe band at 802 cm�1 corresponded to benzene ring vibrations.However, a change in bands was observed following biodegrada-tion due to the formation of metabolites as shown in Fig. 5(b).Here, strong bands appeared at 1460, 1625, 1242 and 884 cm�1

for C–H, C¼O and C–O stretch vibrations and¼C–H out-of-planedeformation vibration. In addition, two new bands at 1737 and3220 cm�1 were also observed, corresponding to ¼C–H, C¼O,and C–O stretch vibrations. The change in bands clearly indicatesthat the formation of metabolites differs from the original sub-strate. Similar results were also observed in a recent study oftriphenylmethane degraded by Staphylococcus epidermidis (Ayedet al., 2009; Ayed et al., 2010).

3.4.4. LC–MS analysis

Liquid chromatography–mass spectrometry analysis was usedto determine degradation products formed in the biodegradationof crystal violet. The LC–MS spectra obtained from samples beforeand after biodegradation for 24 h are presented in Fig. 6(a) and (b),respectively. The peak of crystal violet was observed in Fig. 6(a)at a retention time of 9.0 min before biodegradation. Four newpeaks ( retention time at 8.9 min, 6.7 min, 6.1 min and 5.2 min,respectively) were observed in Fig. 6(b) after degradation, whichshow the formation of four biodegradation products. Basedon fragmentation pattern and m/z values, metabolites wereidentified as of Leucocrystal violet, Michler’s Ketone, [N,N-Dimethylaminophenyl][N-methylaminopheny]benzophenone, andN,N-dimethylaminobenzaldehyde (Fig. 6(b)). Crystal violet was firstbiotransformed into Leucocrystal violet, followed by the asymmetriccleavage by dioxygenase action between carbon of aromatic ringand alkyl carbon resulted in N,N-Dimethylaminophenol andMichler’s Ketone. Then Michler’s Ketone was demethylated to

h. (a) before degradadtion and (b) After degradation. (For interpretation of the

is article.)

Y. Cheng et al. / Ecotoxicology and Environmental Safety 83 (2012) 108–114114

[N,N-dimeth-ylaminophenyl] [N-methylaminophenyl] benzophe-none or reductively split into N,N-dimethylaminobenzaldehydeand N,N-dimethylaminophenol. The result was confirmed by UV–vis spectra and FT-IR spectra.

4. Conclusions

The immobilized B. vietnamiensis C09V on the PVA–alginate–kaolin beads can be used to remove crystal violet from aqueoussolution and this process improves the degradation of crystalviolet. The results shows that 98.6% crystal violet was removedfrom aqueous solution using immobilized cells at pH 5 and 30 1C,while only 94.0% crystal violet was removed by free cells afterdegradation lasting 30 h. Immobilized cells can achieve a higherrate of degradation than free cells, and this is attributable tocrystal violet being adsorbed onto kaolin and following theirdegradation by immobilized cells onto the PVA–alginate–kaolinbeads. This process has been described in various kinetics studies,which show that the adsorption of crystal violet onto thePVA–alginate–kaolin beads used the pseudo-second orderkinetics model and biodegradation of crystal violet was fittedwell by first-order reaction kinetics. However, the removal rateusing immobilized cells was 0.060 mg L�1 h�1, higher than0.044 mg L�1 h�1 for free cells. Results from temperature, con-centrations of crystal violet and the reuse test suggest that theimmobilized B. vietnamiensis C09V on the PVA–alginate–kaolinbeads has the potential to remove crystal violet.

Characterization with SEM shows that cells are successfullyentrapped in PVA–alginate–kaolin gels and biomass was evenlydistributed within the beads, leading to acceleration in the crystalviolet transfer from aqueous solution to immobilized cells. Con-sequently better efficiency in crystal violet removal was obtained.In addition, UV–vis show that the absorption peak at 588 nm wasreduced by the degraded N-bond linkages, Fourier transforminfrared depicted the formation of new bands following thedegradation of crystal violet using alginate–kaolin immobilizedcells. Finally, crystal violet was biodegraded to N,N-dimethylami-nophenol and Michler’s Ketone prior to further degradation ofthese intermediates.

Acknowledgments

The authors sincerely thank Fujian Provincial Developmentand Reform Commission for providing the research funding andFujian Normal University, P. R. China for the supporting from‘‘MinJiang Fellowship’’.

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