Chemosphere 89 (2012) 982–987

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Chemosphere

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Lindane removal by pure and mixed cultures of immobilized actinobacteria

Juliana M. Saez a, Claudia S. Benimeli a,b, María J. Amoroso a,b,c,⇑a Planta Piloto de Procesos Industriales Microbiológicos (PROIMI-CONICET), Avenida Belgrano y Pasaje Caseros, 4000 Tucumán, Argentinab Universidad del Norte Santo Tomás de Aquino (UNSTA), 9 de Julio 165, 4000 Tucumán, Argentinac Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán (UNT), Ayacucho 491, 4000 Tucumán, Argentina

h i g h l i g h t s

" Two entrapped Streptomyces strains showed higher growth compared to free cells." Lindane removal was higher with immobilized cells than with free cells." Cells immobilized in cloth sachets presented highest lindane removal efficiency." Consortia immobilized in cloth sachets allowed 2 additional cycles of 96 h (reuse).

a r t i c l e i n f o

Article history:Received 28 February 2012Received in revised form 23 May 2012Accepted 30 June 2012Available online 26 July 2012

Keywords:LindaneImmobilizationStreptomycesBioremediationMixed culture

0045-6535/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.chemosphere.2012.06.057

⇑ Corresponding author at: Planta Piloto de Procesos(PROIMI-CONICET), Avenida Belgrano y Pasaje CaseroTel.: +54 381 4344888; fax: +54 381 4344887.

E-mail addresses: jsaez@proimi.org.ar (J.M. Saez), cBenimeli), amoroso@proimi.org.ar (M.J. Amoroso).

a b s t r a c t

Lindane (c-HCH) is an organochlorine insecticide that has been widely used in developing countries. It isknown to persist in the environment and can cause serious health problems. One of the strategiesadopted to remove lindane from the environment is bioremediation using microorganisms. Immobilizedcells present advantages over free suspended cells, like their high degradation efficiency and protectionagainst toxins. The aims of this work were: (1) To evaluate the ability of Streptomyces strains immobilizedin four different matrices to remove lindane, (2) To select the support with optimum lindane removal bypure cultures, (3) To assay the selected support with consortia and (4) To evaluate the reusability of theimmobilized cells. Four Streptomyces sp. strains had previously shown their ability to grow in the pres-ence of lindane. Lindane removal by microorganisms immobilized was significantly higher than in freecells. Specifically immobilized cells in cloth sachets showed an improvement of around 25% in lindaneremoval compared to the abiotic control. Three strains showed significantly higher microbial growthwhen they were entrapped in silicone tubes. Strains immobilized in PVA-alginate demonstrated lowestgrowth. Mixed cultures immobilized inside cloth sachets showed no significant enhancement comparedto pure cultures, reaching a maximum removal of 81% after 96 h for consortium I, consisting of the fourimmobilized strains together. Nevertheless, the cells could be reused for two additional cycles of 96 heach, obtaining a maximum removal efficiency of 71.5% when each of the four strains was immobilizedin a separate bag (consortium III).

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction Lindane is a liposoluble compound, which tends to accumulate

The gamma isomer of hexachlorocyclohexane (c-HCH), com-mercially known as lindane, is a highly chlorinated, recalcitrantpesticide, which has been used worldwide as a broad spectruminsecticide for a variety of crops (Phillips et al., 2005). It has alsobeen applied to human health as scabicide and pediculocide, inthe form of lotions and shampoos (Bidlan et al., 2004).

ll rights reserved.

Industriales Microbiológicoss, 4000 Tucumán, Argentina.

benimeli@yahoo.com.ar (C.S.

in fat tissues and as a result biomagnifies in the food chain (Vegaet al., 2007). Chronic exposure to this xenobiotic has been linkedto a range of health effects in humans, including immunosuppres-sion and neurological problems, and it has shown to cause livercancer in rats and mice (Walker et al., 1999). Although nowadaysit has been prohibited in most countries, lindane is still used be-cause of economic reasons and, consequently, new sites are beingcontaminated. Its residues persist in the environment and havebeen found in water, sediments, soil, plants and animals (Con-cha-Graña et al., 2006; Cid et al., 2007; Vega et al., 2007; Carvalhoet al., 2009).

In this context, bioremediation, which involves the use ofmicroorganisms to detoxify and degrade environmental contami-nants, has received considerable attention as an effective

J.M. Saez et al. / Chemosphere 89 (2012) 982–987 983

biotechnological approach to clean up polluted environments.Many microorganisms have been reported to present a great po-tential as pesticide bioremediators. Particularly lindane-degradingbacteria have been isolated and thoroughly studied (Nagata et al.,1999, 2007; Phillips et al., 2005; van Doesburg et al., 2005; Lalet al., 2006; Manickam et al., 2007, 2008).

In previous studies, Benimeli et al. (2003) and Fuentes et al.(2010) isolated and selected wild-type actinobacteria strains,which are able to tolerate and remove lindane from culture mediaand soil. Cuozzo et al. (2009) reported the release of c-2,3,4,5,6-pentachlorocyclohexene and 1,3,4,6-tetrachloro-1,4-cyclohexadi-ene, the first and second products in lindane degradation by Strep-tomyces sp. M7 according to the catabolic pathway proposed byNagata et al. (2007).

A very promising technique that has been gaining special atten-tion during the last years is cell immobilization. This method re-stricts cellular mobility within a defined space, thereby retainingcatalytic activity and enhancing both biological and physical stabil-ity of microorganisms. It can be accomplished by using a variety ofnatural and synthetic matrices, which can alleviate physicochemi-cal challenges, such as pH and temperature (Siripattanakul et al.,2008; Yáñez-Ocampo et al., 2009).

Immobilized cells present several advantages over free sus-pended cells, such as a higher retention of microorganisms in thereactor, enhanced cellular viability (weeks or months), protectionof cells against toxicity, reuse of the cells and easier solid–liquidseparation (Poopal and Laxman, 2009; Ahamad and Kunhi, 2011).Consequently, immobilization of microbial cells and enzymes isincreasingly applied in biotechnological processes for chemicalconversion and bioremediation, obtaining higher biodegradation,higher dilution rate without washout in continuous processesand tolerance against high concentrations of toxic compounds(Wang et al., 2004). Biodegradation with immobilized cells hasbeen assayed for several pesticides, such as diuron (Bazot and Le-beau, 2009), atrazine (Siripattanakul et al., 2009), methyl-para-thion and tetrachlorvinphos (Yáñez-Ocampo et al., 2011) andpentachlorophenol (León-Santiestebán et al., 2011). However, noinformation is available on the use of immobilized actinobacteriafor lindane removal. Therefore, the present work aimed to assessthe ability of four isolated actinobacteria to remove lindane froma liquid medium using four different immobilization techniques.The matrix with highest pesticide removal was selected and fur-ther assayed using a mixed culture of the studied actinobacteria.

2. Materials and methods

2.1. Reactive agents

Lindane (99% pure), polyvinyl alcohol (PVA) (80% hydrolyzed,molecular weight 9000–10,000) and sodium alginate (medium vis-cosity) were purchased from Sigma–Aldrich Co. (St. Louis, MO,USA). All other chemicals used throughout this study were of ana-lytical grade and were obtained from standard manufacturers.

2.2. Microorganisms and culture media

Four Streptomyces sp. strains (Streptomyces sp. A2, A5, A11 andM7) were used in this study. It were previously isolated fromtwo different contaminated environments in Argentina (Benimeliet al., 2003; Fuentes et al., 2010) and then selected for their abili-ties to grow in the presence of lindane as carbon source and to re-move it from the culture medium, as pure and mixed cultures(Fuentes et al., 2011). The strains were cultured on Starch-Casein(SC) plates, containing in g L�1: starch, 10.0; casein, 1.0; K2HPO4,0.5; agar, 15.0 (Hopwood et al., 1985). The pH was adjusted to

7.0 prior to sterilization by autoclaving at 121 �C for 15 min. Plateswere incubated at 30 �C for 7 d.

Tryptic Soy Broth (TSB) was used for the inocula preparation.The pH was adjusted to 7.3 ± 0.2 and the medium was then steril-ized by autoclaving at 121 �C for 15 min.

All removal assays were carried out in Minimal Medium (MM),which contained in g L�1: L-asparagine, 0.5; K2HPO4, 0.5;MgSO4�7H2O, 0.20; FeSO4�7H2O, 0.01 (Hopwood et al., 1985). ThepH of the medium was adjusted to 7.0 prior to sterilization byautoclaving (121 �C, 15 min) and finally the medium was supple-mented with 1.66 mg L�1 of lindane as carbon source.

2.3. Preparation of the inocula

The four actinobacteria (Streptomyces sp. A2, A5, A11 and M7)were individually cultured in TSB and incubated at 30 �C for 72 hon a shaker (200 rpm). Then, the biomass pellets were harvestedby centrifugation at 10,000 rpm and washed with sterile distilledwater for their use in the subsequent immobilization assays.

2.4. Immobilization assays

Each strain was individually immobilized in four matrices: agarcubes, PVA-alginate beads, silicone tubes and cloth sachets. All theequipments and materials used in the preparation of the entrappedcells were sterilized and the operations were carried out understerile conditions.

2.4.1. Immobilization in agar5 mL of fluid agar (3%) containing the corresponding Streptomy-

ces sp. strain at a final concentration of 7.5% (w/v) (wet weight)were poured into a 50 mm diameter Petri dishes. Agar plates werekept at room temperature for solidification and afterwards the agarblocks were cut into 0.5 � 0.5 cm cubes using a sterile scalpel.

2.4.2. Immobilization in PVA-alginate5 mL of a mixture of 5% (w/v) PVA, 0.8% (w/v) calcium alginate

and 7.5% (w/v) (wet weight) of the corresponding Streptomyces sp.strain were poured into a stirred solution of 2% CaCl2�2H2O with a1000 lL-micropipette and incubated at room temperature for 1 h.Then the beads (3–5 mm diameter) were washed three times withsterile distilled water (Pattanapipitpaisal et al., 2001).

2.4.3. Immobilization in silicone tubesA sterile 9-cm-long silicone tube (inner diameter 3 mm, wall

size 1 mm) was injected with a fluid mixture of SC medium (pre-pared with 40 g L�1 of agar) containing the corresponding Strepto-myces sp. strain at a final concentration of 7.5% (w/v) (wet weight).When the mixture was solidified, the tube was cut into pieces ofapproximately 1 cm long. The silicone tubes remained open at bothends (Boon et al., 2002; Mertens et al., 2006).

2.4.4. Immobilization in cloth sachetsCloth sachets (2.5 cm � 3.5 cm) were prepared with voile and

sterilized in autoclave. Then they were aseptically loaded withthe same inoculum quantity of each Streptomyces strain used inthe other immobilization techniques (Srivastava and Kundu, 1998).

2.5. Bacterial growth and lindane removal in pure cultures

Individually immobilized Streptomyces strains were cultured inthe presence of lindane. Therefore, the agar cubes obtained fromone Petri dish, 5 g of beads, nine pieces of 1-cm-long silicone tubeand one sachet were placed in each case in a 250 mL-Erlenmeyercontaining 100 mL of MM supplemented with 1.66 mg L�1 of lin-dane and incubated at 30 �C on a shaker (200 rpm) for 96 h. After

A2 A5 A11 M7

0.0

5.0x105

1.0x106

1.5x106

2.0x106

1.0x107

2.0x107

3.0x107

4.0x107

5.0x107

Mic

robi

al g

row

th (

CF

U g

-1)

Strains

Free cells

Agar cubes

PVA-alginate beads

Silicone tubes

Cloth sachets

Fig. 1. Microbial growth (CFU g�1) of free and immobilized pure cultures ofStreptomyces sp. A2, A5, A11 and M7. Error bars indicate standard deviations. Barsshowing one, two and three asterisks indicate they were significantly different fromthe others and among them (P 6 0.05, Tukey post-test).

984 J.M. Saez et al. / Chemosphere 89 (2012) 982–987

that, the entrapped cells were collected to assess microbial growthby determination of the colony forming units per gram of support(CFU g�1) and supernatant samples were taken to determine resid-ual lindane concentration by gas chromatography with an electronmicro-capture detector (GC-lECD). The same assays were carriedout with free cells. MM containing the different immobilizationmatrices without microorganisms was used as control.

2.6. Gas chromatography

The residual lindane was extracted from cell-free supernatants(10,000 rpm, 30 min, 4 �C) by solid phase extraction (SPE) using aC18 column (Agilent, Lake Forest, USA).

Extracts were quantified in an Agilent 7890A Gas Chromato-graph equipped with a HP5 capillary column (30 m � 0.53 mm �0.35 m) and 63Ni lECD detector, a split/splitless Agilent 7693Binjector and Agilent ChemStation software. Quantitative sampleanalysis was performed using appropriate calibration standards(AccuStandard).

2.7. Bacterial growth and lindane removal by mixed cultures

Based on the results in Section 2.5, the support with highest lin-dane removal was selected and subsequently assayed with mixedcultures of the four studied Streptomyces sp. in three differentways: (I) The four immobilized Streptomyces sp. together, (II)Two immobilized and two free strains, and (III). Each strain indi-vidually immobilized. Each of the three consortia had at a finalconcentration of 7.5% (w/v) (wet weight) and was cultured inMM with 1.66 mg L�1 of lindane and incubated for 96 h at 30 �C.Samples were collected every 24 h to analyse residual lindaneand microbial growth inside the support. Biomass of cells releasedfrom the support was also determined. These parameters weremeasured by using the methodology previously described (Sec-tions 2.5 and 2.6). Afterwards, the reusability of the immobilizedconsortia was tested in two additional cycles of 96 h each. Residuallindane was examined at the end of each period.

2.8. Lindane removal performance

The lindane removal performance was evaluated according tothe efficiency (E) and the removal rate (Rr), which were calculatedas follows:

Eð%Þ ¼ ðLi � Lf ÞLi

� 100%

RrðmgL�1 h�1Þ ¼ Li � Lf

tf � ti

Li is the lindane concentration (mg L�1) at time ti, Lf is the residuallindane concentration (mg L�1) at time tf, ti is the initial time of theexperiment (t = 0 h), tf is the total incubation time (h).

2.9. Statistical analyses

All experiments were carried out in triplicate and the results areexpressed as average values. A two-way ANOVA was used for var-iance analysis (Tukey post-test, P 6 0.05). All statistical analyseswere performed using a professional version of Infostat software.

3. Results and discussion

3.1. Microbial growth in pure cultures of Streptomyces sp

Immobilized microbial systems have shown several advantagesover free cells, such as a higher degradation rate, enhanced toler-

ance to high concentrations of toxic compounds and the possibilityof reusing the cells. The present study intended to assess the per-formance of four previously isolated Streptomyces strains in the re-moval of lindane from liquid batch systems with free andimmobilized cells. Four different matrices were assayed: agarcubes, PVA-alginate beads, silicone tubes and cloth sachets.

Microbial growth of pure cultures is shown in Fig. 1. The fourStreptomyces sp. strains (A2, A5, A11 and M7) were able to growin MM supplemented with lindane as carbon and energy source.All strains showed lowest growth when immobilized in PVA-algi-nate beads compared to the other three supports. Polyvinyl alcohol(PVA) is a cheap synthetic organic polymer, which is widely usedfor cell immobilization, mainly combined with alginate, becauseof its high mechanical strength, durability and its innocuousnessto both microorganisms and environment. However, cells mayhave experienced physical stress during the PVA-alginate immobi-lization process or may have suffered low oxygen and/or substratediffusion due to the matrix. In a previous study, Siripattanakulet al. (2008) reported a lower proportion of viable cells underimmobilized conditions with phosphorylated PVA as support com-pared to free cells, indicating that immobilization produced a neg-ative effect on cell viability.

Streptomyces sp. A11 and M7 presented maximum growth inthe four immobilized forms. A possible explanation is that the sup-port would be acting as a protection for the cells against the toxic-ity of lindane in the medium. However, there is no clearunderstanding of how immobilization processes protect microor-ganisms (Boon et al., 2002).

Streptomyces sp. A2, A5 and A11 showed significantly highergrowth values (P 6 0.05) when they were entrapped in siliconetubes compared to the other immobilization techniques and freecells. This could be due to an adequate diffusion of both oxygenand substrates provided by the medium through the agar in the sil-icone tubes, since both ends were open. Moreover, the tubes con-tained nutritive agar inside, absent in the other immobilizationmodes, which may have helped sustain cell growth. Streptomycessp. A11 immobilized in agar cubes and cloth sachets presented sig-nificantly different growth values (P 6 0.05) compared with bead-immobilized and free cells. In contrast, Streptomyces sp. M7 showedno significant differences among the four immobilizationtechniques.

3.2. Lindane removal by pure Streptomyces sp. cultures

Lindane removal efficiency and microbial growth in Streptomy-ces sp. A11 and M7 in the four studied matrices was significantly

Table 1Lindane removal rate (mg L�1 h�1) of pure and mixed cultures ofthe four Streptomyces sp. strains assayed and immobilized in clothsachets, during three consecutive cycles of 96 h each.

Immobilized Streptomycesstrains

Removal rate(mg L�1 h�1)

Pure culturesA2 0.0179 ± 0.0003ef

A5 0.0182 ± 0.0004f

A11 0.0188 ± 0.0004f

M7 0.0179 ± 0.0003ef

Mixed cultures

Mix I1st Use 0.0169 ± 0.0005def

1st Reuse 0.0123 ± 0.0017c

2nd Reuse 0.0123 ± 0.0014c

Mix II1st Use 0.0127 ± 0.0024c

1st Reuse 0.0079 ± 0.0011b

2nd Reuse 0.0035 ± 0.0024a

Mix III1st Use 0.0169 ± 0.0001def

1st Reuse 0.0149 ± 0.0002cde

2nd Reuse 0.0142 ± 0.0006cd

Mix I, II and III refer to Mixed Cultures I, II and III, respectively(see Section 2.7 for details). Different letters indicate significantdifferences (P 6 0.05, Tukey post-test).

J.M. Saez et al. / Chemosphere 89 (2012) 982–987 985

higher (P 6 0.05) than in free cells, which means that these strainswere metabolically more active when they were immobilized thanwhen they were free. Other authors have also observed increasedmetabolic activity with encapsulated cells (Mertens et al., 2006;Siripattanakul et al., 2008), but the underlying mechanism hasnot yet elucidated.

Regarding the different matrices, lindane removal was mostefficient for all strains when immobilized in silicone tubes followedby cloth sachets (Fig. 2). However, since the lindane removal effi-ciency of the abiotic silicone tube control (86.5%) was not signifi-cantly different from the strains immobilized in this support(92–95%), most removal was likely caused by adsorption of c-HCH to the tubes. Mertens et al. (2006) obtained similar resultswhen they estimated the amount of c-HCH that could be adsorbedto agar-filled silicone tubes used for encapsulation, revealing thatthe tubes were responsible for the removal of a high percentageof the initially spiked lindane.

In contrast, when Streptomyces sp. strains were immobilized incloth bags, lindane removal improved in approximately 25% (85–90%) compared to the abiotic control (63%), although pesticideadsorption contributed too. The difference with all the immobi-lized strains was significant (P 6 0.05), evidencing microbial ac-tion. As a result, this support was selected to evaluate thelindane removal ability of mixed cultures. The removal rate wasalso calculated for pure cultures immobilized in this support, andpartial differences in this parameter could be observed amongsome of the strains (Table 1).

Several researchers have demonstrated that immobilization en-hanced tolerance to toxic compounds and the degrading ability ofthe cells. Our results with cells immobilized in agar and PVA-algi-nate did not show highest lindane removal, whereas Ahamad andKunhi (2011) reported that Pseudomonas sp. CP4 remarkably im-proved the removal of phenol when it was immobilized in Ca-algi-nate and agar beads, and removal by agar-entrapped cells was farsuperior to Ca-alginate-immobilized microorganisms. Howeverother researchers found that calcium alginate was a better matrixthan agar–agar regarding degradation activity (Dey and Roy, 2009).

3.3. Microbial growth and lindane removal by mixed cultures ofStreptomyces sp

A mixed culture composed of the four strains assayed was en-trapped in cloth sachets in three different ways. (I) The four actino-bacteria were immobilized together in a cloth sachet, (II)Streptomyces sp. A5 and A11 were immobilized together in a bagand Streptomyces sp. A2 and M7 were kept free (based on the high-

Abiotic control A2 A5 A11 M70

10

20

30

40

50

60

70

80

90

100

Lin

dane

rem

oval

eff

icie

ncy

(%)

Strains

Free cells Agar cubes PVA-alginate beads Silicone tubes Cloth sachets

Fig. 2. Lindane removal efficiency (%) of free and immobilized pure cultures ofStreptomyces sp. A2, A5, A11 and M7. Error bars indicate standard deviations. Barsshowing one asterisk indicate they were significantly different from the others. Thebar showing two asterisks indicates it presented significant differences with thebars showing three asterisks (P 6 0.05, Tukey post-test).

er percentages of lindane removal showed in Section 3.2), and III:Each strain was individually immobilized in a cloth bag; i.e. fourbags with one strain each.

In consortium I, microbial growth of cells immobilized in the sa-chet was similar to that by Streptomyces sp. A5, A11 and M7 whenthey were individually immobilized in the same matrix. The bio-mass of the cells released from the support was also examined asan inverse measure of the stability of the immobilization and ma-trix resistance (Fig. 3A). After 96 h, free cells represented only0.12% of the cells grown inside the bag and 1.2% of the initial bio-mass, revealing that the immobilization technique was efficientand the support did not suffer any disintegration. There was no sig-nificant difference between the mixed culture and the pure cul-tures immobilized in the cloth bags regarding lindane removal,and maximum removal was 81% after 96 h; nevertheless it is ac-cepted that the metabolic synergism among different species orstrains can promote the degradation of recalcitrant moleculesand in fact, this process can be further enhanced by cellular immo-bilization (Yáñez-Ocampo et al., 2009). On the other hand, the sa-chets could be reused for two additional culture cycles of 96 h,with a removal efficiency of 59% in each case (Fig. 4). The lindaneremoval rate is shown in Table 1. The first use of mixed culture Ipartially differed from the removal rate by pure cultures immobi-lized in the same support, while the removal rate of reused bagswas significantly lower (P 6 0.05).

Mixed culture II consisted of 50% immobilized and 50% freecells. The first 24 h showed an important decrease in the biomassof the free cells (Fig. 3B), which could be due to adsorption of thecells to the outer side of the bag. However, this physical phenom-enon is apparently reversible because in the next hours the bio-mass of free cells increased again and remained practically stableuntil 96 h. Cells grown inside the bag presented values similar tothose obtained in consortium I, while the lindane removal waslower (71%). Lindane removal efficiency of the reused cells was sig-nificantly lower (P 6 0.05) in mixed culture II than in consortium I,reaching 40.5% removal in the first reuse cycle and 17.8% in thesecond (Fig. 4). The removal rate of consortium II was significantlylower (P 6 0.05) than the pure cultures and mixed culture I(Table 1).

0 20 40 60 80 100

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Time (h)

Res

idua

l Lin

dane

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)

02x1044x10 46x1048x1041x1051x1051x1052x1052x105

1x1072x1073x1074x1075x1076x1077x1078x107

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02x1044x1046x1048x104

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0 20 40 60 80 1000

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1x1042x1043x1044x1045x104

1x107

2x107

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Mic

robi

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-1)

C

Mic

robi

al g

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th (

CF

U g

-1)

Mic

robi

al g

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th (

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U g

-1)

Fig. 3. A, B and C. Residual lindane percentages (dashed line) and microbial growth(solid line) of the mixed cultures I, II and III, respectively, immobilized in clothsachets. Immobilized cells; Free cells; Immobilized cells;

Abiotic control. Error bars indicate standard deviations.

IIIIII0

10

20

30

40

50

60

70

80

90

100

Lin

dane

rem

oval

eff

icie

ncy

(%)

Consortia

1st Reuse cycle

2nd Reuse cycle

a

b

c c

Fig. 4. Lindane removal efficiency (%) of the reused cells of the consortia I, II and IIIimmobilized in cloth sachets for two additional cycles of 96 h each. Error barsindicate standard deviations. Different letters indicate there were significantdifferences (P 6 0.05, Tukey post-test).

986 J.M. Saez et al. / Chemosphere 89 (2012) 982–987

Consortium III demonstrated lower microbial cell growth thanthe other two consortia, with a final value of 3.8 � 107 CFU g�1

(Fig. 3C). A small proportion of the cells inoculated inside the bagswas released from them, representing after 96 h only 0.1% of thecells grown inside the sachet and 0.28% of the initial inoculum. Lin-dane removal showed almost no difference with the abiotic control(Fig. 3C), although reuse of the cells presented promising resultswith this consortium, obtaining a removal efficiency of 71.5% and68% in the first and second reuse of the immobilized cells, respec-tively (Fig. 4), and a removal rate of 0.0149 ± 0.0002 for the firstand 0.0142 ± 0.0006 for the second reuse cycle, showing partial dif-ferences with the first use (Table 1).

Other studies have reported that cells entrapped in calciumalginate could be used five times and agar–agar entrapped cellsthree times (Dey and Roy, 2009). Nigam et al. (2009) informed that

they reused agar-immobilized Streptomyces sp. 20 times withoutmuch loss of activity. Our study assessed the reusability of the cellsduring three periods of 96 h each, but further studies about the re-use of the immobilized cells are necessary.

4. Conclusions

The use of immobilized indigenous actinobacteria is a promis-ing alternative to bioremediation of polluted sites. The advantagesof immobilized Streptomyces strains make them suitable candi-dates for the development of techniques for lindane removal fromcontaminated environments. Cells immobilized in cloth sachetsshowed the best behavior. Therefore, this material may be anattractive support for microbial cell entrapment. However, moreelaborated studies with respect to the longevity and reusabilityof the immobilized cells will have to be conducted.

Acknowledgements

This work was financially supported by Consejo de Investigaci-ones de la Universidad Nacional de Tucumán (CIUNT), AgenciaNacional de Promoción Científica y Tecnológica (ANPCyT) andConsejo Nacional de Investigaciones Científicas y Técnicas (CONI-CET). The authors gratefully acknowledge Dr. A. Álvarez for hercontribution to statistical analysis and Mr. G. Borchia for his tech-nical assistance.

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