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Journal of Biotechnology 157 (2012) 148– 153

Contents lists available at SciVerse ScienceDirect

Journal of Biotechnology

j ourna l ho me pag e: www.elsev ier .com/ locate / jb io tec

mmobilization of Delftia tsuruhatensis in macro-porous cellulose andiodegradation of phenolic compounds in repeated batch process

. Juarez Jimeneza, P. Reboleiro Rivasa, J. Gonzalez Lopeza, C. Pesciarolib, P. Barghinib, M. Feniceb,c,∗

Instituto del Agua, University of Granada, Granada, SpainDEB: Dipartimento di Scienze Ecologiche e Biologiche, University of Tuscia, Viterbo, ItalyLaboratory of Applied Marine Microbiology, Conisma, University of Tuscia, Viterbo, Italy

r t i c l e i n f o

rticle history:eceived 1 August 2011eceived in revised form9 September 2011ccepted 22 September 2011vailable online 1 October 2011

eywords:

a b s t r a c t

Delftia tsuruhatensis BM90, previously isolated from Tyrrhenian Sea and selected for its ability to degradea wide array of phenolic compounds, was immobilized in chemically modified macro porous cellulose.The development of bacterial adhesion on the selected carrier was monitored by scanning electronmicroscopy. Evident colonization started already after 8 h of incubation. After 72 h, almost all the car-rier surface was covered by the bacterial cells. Extracellular bacterial structures, such as pili or fimbriae,contributed to carrier colonization and cell attachment. Immobilized cells of D. tsuruhatensis were testedfor their ability to biodegrade a pool of 20 phenols in repeated batch process. During the first activation

mmobilizationepeated batch processelftia tsuruhatensisolyphenols degradation

batch (72 h), 90% of phenols degradation was obtained already in 48 h. In the subsequent batches (upto 360 h), same degradation was obtained after 24 h only. By contrast, free cells were slower: to obtainalmost same degradation, 48 h were needed. Thus, process productivity, achieved by the immobilizedcells, was double than that of free cells. Specific activity was also higher suggesting that the use of immo-bilized D. tsuruhatensis BM90 could be considered very promising in order to obtain an efficient reusablebiocatalyst for long-term treatment of phenols containing effluents.

. Introduction

Phenolic compounds are present in effluents from several indus-ries such as coal conversion (Lu et al., 2009), wine and olive oilroduction (Cerezo et al., 2008; Cerrone et al., 2011), pharma-eutical (Nalewajko-Sieliwonjuk et al., 2008), textile and paperanufacturing (Annachhatre and Gheewala, 1996), brewing, man-

facture of resins, chemicals and oil (Abrahamssom and Klick,991). These compounds are considered as priority environmen-al pollutants because of their high toxicity at low concentrationsKallel et al., 2009). In particular, the removal of toxic phenols,rom different wastewater typologies, is an important issue to beddressed. In fact, their presence in water and soil has become aignificant pollution problem. Thus, many efforts, including variousiological treatments, have been made in order to reduce phenoloncentration in various industrial and agro-industrial effluentsHamdi and García, 1993).

However, biological treatment of phenol-containing wastew-ter is often possible only after effluents dilution, due to theicrobial growth inhibition by phenols (Quaratino et al., 2007).

∗ Corresponding author at: DEB: Dipartimento di Scienze Ecologiche e Biologiche,niversity of Tuscia, Viterbo, Italy. Tel.: +39 0761 357318.

E-mail address: fenice@unitus.it (M. Fenice).

168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2011.09.026

© 2011 Elsevier B.V. All rights reserved.

Various phenols-degrading microorganisms have been isolatedand evaluated for the control of phenol pollution (Hamdi andGarcía, 1993; Quaratino et al., 2007; Juarez-Jimenez et al., 2010)but the knowledge on new biotechnologies is still insufficient.Among them, the following microorganisms seem to be particularlypromising: Alcaligenes sp. (Valenzuela et al., 1997), Azotobacter sp.(Wieser et al., 1994, Juarez et al., 2005), Cryptococcus sp. (Morsenand Rehm, 1987), Panus tigrinus (Quaratino et al., 2007), Phane-rochaere sp. (Perez et al., 1997), Pseudomonas sp. (Chitra et al., 1995)and Rhodococcus sp. (Oh and Han, 1997). These microorganismshave been selected for their ability to use phenolic compounds assole carbon source.

Delftia tsuruhatensis is a Gram-negative rod showing highlypromising metabolic diversity for the biodegradation of variousrecalcitrant compounds. Its degrading ability has been demon-strated for anilines (Liang et al., 2005; Sheludchenko et al., 2005;Zhang et al., 2010), terephthalate, protocatechuate (Shigematsu etal., 2003), and (S)-2,2-dimethylcyclopropanecarboxamide (Zhenget al., 2007). In addition, strain BM90 of D. tsuruhatensis wasrecently studied for its capacity to degrade a pool of mixed phenols(Juarez-Jimenez et al., 2010).

In general, all the microorganisms studied for their phenolsdegradation abilities had been used in free form but the obtainmentof a reusable biocatalyst for long-term biodegradation processeswould be a winning strategy (Wang et al., 2010).

B. Juarez Jimenez et al. / Journal of Biotechnology 157 (2012) 148– 153 149

Table 1Physico-chemical specifications of different macroporous chemically modified cellulose carriers.

Carrier Chemical modification Electric charge Shape Pore size (�m)

BR-L01T70 Poliethyleneimine (no PEI elution) Low Cubic 70b

BR-L01T500 Poliethyleneimine (no PEI elution) Low Cubic 500a

BZ-H01B70 Poliethyleneimine High Bead 70b

BZ-H01T500 Poliethyleneimine High Cubic 500a

CM-L01T70 Carboxymethyl group Low Cubic 70b

CM-L01T500 Carboxymethyl group Low Cubic 500a

FN-S01B70 Non-electric charged Non-modified Bead 70b

Carrier properties: Average size of the carrier, 1 mm; absorption capacity, 26.3 mL g−1; temperature stability, 130–140 ◦C; real cellulose density, 1.45–1.55 g mL−1 (data fromM

eemmlibip(tai

bdb2saa

gtr

Blopilc

2

2

sobE7C

2

s

Carrier beads were fixed for 2 h with 2.5% glutaraldehyde in0.1 M cacodylate buffer at pH 7.0, and then rinsed and treated

icrobe technical sheets, Biomaterial Co. Ltd., Japan).a Determined as size of Na2SO4

− .b Determined by mercury porosimeter.

Immobilization is one of the most successful technologies toxtend biocatalysts operational lifetime allowing their easy recov-ry from the reaction mixture (Wang et al., 2010). Immobilized cellsaintain their biological functions with increased stability thatay often lead to increased or improved cell activities. Immobi-

ization is particularly suitable when microbial cells are employedn domestic and industrial wastewater treatments generatingiofilms to be used in packed-bed (submerged filters) and/or flu-

dized bed reactors (Gómez-Silván et al., 2010). In addition, they arearticularly useful in semi-continuous and/or continuous processesFenice et al., 1998) and are mainly suitable for large scale indus-rial applications. Cells may be immobilized by various physicalnd chemical methods and carrier selection is always an importantssue for process success (Fenice et al., 1998).

D. tsuruhatensis, immobilized on polycaproamide, fibers haseen successfully used in batch and continuous processes toegrade aniline (Sheludchenko et al., 2005). In addition, it haseen encapsulated in Na-alginate for the degradation of (R)-,2-dimethylcyclopropane carboxamide showing good operationaltability (Wang et al., 2010). Also, co-immobilization of Delftia sp.nd other bacteria are reported (Bazot et al., 2007; De Gusseme etl., 2011).

However, no extensive studies have been carried out to investi-ate the metabolic capacity of immobilized bacteria for their abilityo metabolise phenolic compounds and no work regards D. tsu-uhatensis.

In this work we studied the immobilization of D. tsuruhatensisM90 on different carriers (chemically modified macro porous cel-

uloses) following the bacterial adhesion on a selected support bybservations under the scanning electron microscope (SEM). Then,olyphenols degradation by the immobilized cells was carried out

n shaken cultures, repeated-batch processes, in order to studyong-term cells reusability for possible applications with phenol-ontaining effluents.

. Materials and methods

.1. Microorganism

Delftia tsuruhatensis BM90 was previously isolated from wateramples collected at 90 m depth in the Tyrrhenian Sea off the coastf Giglio Island, Grosseto, Italy (Fenice et al., 2007). The strain haseen deposited to the Spanish Type Culture Collection (Colecciónspanola de Cultivos Tipo, CECT) with the access number: CECT427. During the study, strain BM90 was maintained at 4 ◦C on Plateount Agar slants (PCA, Oxoid, UK) and routinely sub-cultured.

.2. Media and culture conditions

Starter cultures of BM90 in shaken flasks, obtained from PCAlants, were incubated on a rotary shaker at 28 ◦C at 180 rpm in

nutrient broth (NB) (Difco, USA). After 18 h of incubation, cellswere harvested by centrifugation, washed twice and resuspendedin sterile phosphate buffer (pH 7.0) to obtain the requested finalconcentration (106 cells mL−1) for the inoculation of further shakencultures. The necessary amount of cell suspension was added toErlenmeyer flasks containing 50 mL of the following media.

Carrier selection medium (CM): 1000 mg L−1 of yeast nitro-gen base (YNB, Difco, USA). Repeated-batch medium (RM):670 mg L−1 YNB amended with 500 mg L−1 of a pool of the follow-ing 20 phenolic compounds (25 mg L−1, each): 3-hydroxybenzoicacid, 4-hydroxybenzoic acid, veratric acid, vanillinic acid, gen-tisic acid, protocatechuic acid, siringic acid; p-coumaric acid,ferulic acid, caffeic acid, siringaldehyde, protocatechuic alde-hyde, vanillin, 4-hydroxybenzaldehyde; 3-hydroxyphenylacetic,4-hydroxyphenylacetic acid, 3,4-dihydroxyphenylacetic acid, cat-echol, 4-methilcatechol, 3-phydroxyphenylpropionic acid (Juarez-Jimenez et al., 2010). All phenolic compounds were from Fluka withHPLC grade. Media were filter-sterilised (Millipore, 0.22 �m) andpH adjusted to 7.0 using sterile 0.1 N NaOH.

2.3. Carrier selection and immobilization procedure

The carriers used in this study were particles of the follow-ing chemically modified macro-porous cellulose (MPC) (Microcube,Biomaterial Co. Ltd., Fukui, Japan): BZ-H01B70, BR-L01T70, FN-S01B70, CM-L01T70, CM-L01T500, BR-L01T500 and BZ-H01T500(see Table 1 for carrier physico-chemical specifications). Carrierswere sterilised at 121 ◦C for 20 min.

For carrier selection, 20% (w/v) of each macro-porous cellulosewas suspended in 50 ml of CM (in 250 mL Erlenmeyer flasks) andinoculated with the above described cell suspension to obtain thefinal concentration of 106 cells mL−1. Flasks were then incubatedfor 72 h at 28 ◦C and 50 rpm to promote cell adhesion. In orderto evaluate immobilized cell retention and leakage by the carrier,the colonized supports have been extensively washed in phosphatebuffer (pH 7.0) and incubated for further 48 h at 28 ◦C and 180 rpm.

Free and immobilized biomass was measured by dry weightdeterminations.

Development of the bacterial adhesion to the carrier selected forthe repeated batch processes was followed by SEM observations.

2.4. Biological samples preparation for Scanning ElectronMicroscopy

with 1% osmium-oxide for 3 h. After dehydration in an ethanolseries (30, 50, 70, 90% and absolute), beads were critical point driedand mounted on support stubs. A Hitachi S-510 scanning electronmicroscope was used for samples observation without gold coating.

150 B. Juarez Jimenez et al. / Journal of Biotechnology 157 (2012) 148– 153

Table 2Immobilization of Delftia tsuruhatensis BM90 on different carriers after 72 h of incubation in CM and further 48 h in phosphate buffer.

BR-01T70 BR-L01T500 BZ-01B70 BZ-H01T500 CM-01T70 CM-01T500 FN-S01B70

IB 0.54a ± 0.072 0.26b ± 0.028 0.25b ± 0.062 0.20b ± 0.046 0.19b ± 0.036 0.32bc ± 0.041 0.39c ± 0.023FB 0.14a ± 0.010 0.08b ± 0.001 0.07b ± 0.018 0.29d ± 0.021 0.05b ± 0.007 0.10c ± 0.012 0.13ac ± 0.013TB 0.68a ± 0.082 0.34b ± 0.029 0.32b ± 0.080 0.49c ± 0.067 0.24b ± 0.043 0.42bc ± 0.053 0.52ac ± 0.036I/F 3.82 3.31 3.41 0.70 3.6 3.16 2.96RB 98.3 96.5 98.8 91.2 65.8 76.5 58.1

I L−1) aC ned imL (P < 00

2

Cbta(rnwwircup

wb

2

cFtai

2

cs

3

3

i(camMeu2

ftl

B, immobilized biomass (g L−1) after 72 h of incubation in CM; FB, free biomass (gM; I/F, immobilized/free biomass rate after 72 h of incubation in CM and RB, retaiine means followed by the same superscript letter were not significantly different

.5. Repeated-batch cultivations

Processes were carried out both with free and immobilized cells.ell immobilization was carried out as reported above. After immo-ilization, beads were washed twice with sterile distilled watero remove any residual carbon source, added with 50 mL of RMnd incubated on a rotary shaker at 180 rpm and 28 ◦C for 72 hactivation batch). Repeated-batch cultivation were carried outemoving the exhausted medium at the time of maximum phe-ol degradation and, after washing the beads, with sterile distilledater, adding 50 ml of fresh RM (Fenice et al., 1998). Experimentsere carried out in triplicate. Two controls were carried out: non-

noculated carrier beads were added with RM and submitted to theepeated batch process to detect possible phenols absorption to thearrier (C1) and non-inoculated RM without carrier was incubatednder shaken condition for the entire process (360 h) to detectossible abiotic phenols degradation (C2).

Biomass of free and immobilized cells was measured by dryeight determinations at the beginning and the end of the repeated

atch process.

.6. Analytical assays

Two different methods were used to determine the con-entration of phenolic compounds in the culture media. Theolin–Ciocalteau method was used to measure total phenols con-ent as reported by Singleton et al. (1999). Gas Chromatographicnalysis (GC–MS) was used to determine concentration of the var-ous phenols according to Juarez-Jimenez et al. (2010).

.7. Statistical analysis

One-way analysis of variance (ANOVA) and pairwise multipleomparison procedures (Turkey test) were carried out using thetatistical software SigmaStat (Jandel, San Rafael, CA).

. Results and discussion

.1. Carrier selection

Macro-porous cellulose has been reported as one of the mostnteresting non-conventional materials for cell immobilizationFenice et al., 1998): its use, in fact, could overcome some typi-al limitations of other materials (e.g. alginate, carrageenan, agarnd glass beads) that often present technical problem such as lowechanical stability or low oxygen permeation rate (Pörtner andärkl, 1996). However, its use is still very scarcely diffused and

ven the most recent attempts to immobilize Delftia cells were donesing different supports (Sheludchenko et al., 2005; Bazot et al.,007; Wang et al., 2010; De Gusseme et al., 2011)

Criterion for MPC selection was the affinity of the bacterial cellsor the carrier, measured in terms of cell attachment and reten-ion, leading to good colonization of carrier surface, minimal celleakage from the carrier and, as a consequence, least presence of

fter 72 h of incubation in CM; TB1, total biomass (g L−1) after 72 h of incubation inmobilized biomass (% of IB) after incubation for further 48 h in phosphate buffer.1) as determined by the Tukey test.

freely growing microorganisms (Leenen et al., 1996; Quiros et al.,1996; Fenice et al., 1998). Thus, MCPs were preliminarily tested inorder to select the most suitable support for D. tsuruhatensis BM90immobilization (Table 2).

Not all the carriers supported same cell adhesion and, in somecases, growth of free cells was particularly evident as indicated bythe immobilized/free (I/F) cell rate. In case of BZ-H01T500, free cellgrowth predominated. As for cell leakage from the colonized beads,all “BR” and “BZ” supports showed high levels of retainde biomass(RB ≥ 91.2%). By contrast performance of the other carriers was def-initely lower (RB not exceeding 76.5%). Lowest cell leakage wasrecorded for BZ-01B70 (RB = 98.8%) that, however, showed ratherlow immobilized biomass (IB = 0.25 ± 0.062 g L−1).

Best carrier colonization (IB = 0.542 ± 0.072 g L−1), highest I/Frate (3.82) and very good RB (98.3%) were recorded for BR-L01T70that was selected for the repeated-batch processes. Actually, a goodI/F rate was recorded also for other supports but with definitelylower immobilized biomass.

It is worth nothing that this support appears particularlyappropriate also for other microbial cell adhesion as alreadydemonstrated for fungal immobilization by the work of Fenice et al.(1998).

3.2. Development of the immobilized bacterial cells on theselected carrier

SEM observations of Microcube BR-L01T70 during the immo-bilization process confirmed very good and progressive supportcolonization (Fig. 1).

As expected, cell adhesion after the inoculum (0 h) was almostnegligible. By contrast, after 8 h of incubation, carrier colonizationwas already evident and cell adhesion to the support progressivelyincreased during the following hours to reach a very good surfacecoverage at the 72 h.

As for the mechanism of the bacterial cells adhesion to carriersurface, possibly, due to the electric charge of the modified cellu-lose, some nonspecific absorption occurred. However, apparentlyD. tsuruhatensis BM90 actively attached to the carrier by the pro-duction of specific structures (Fig. 2A–C). Most of the cells appearedstrongly connected with the carrier by one or several fimbria-likeappendixes. Although during SEM samples preparation artefactscould occur, most of the cells showed these structures.

Intense bacterial growth (cell division) was recorded for theattached cells also (Fig. 2A, E and F), thus carrier colonization couldoccur by attachment of free living cells but also by the alreadyattached bacteria. The structure of the attached biomass appearedto be rather complex with contacts among different cells occurringby formation of pili (Fig. 2B). It is known that there is a connec-tion between surface colonization and bacterial communicationby pili. This mechanism involves also conjugative plasmids induc-

ing planktonic bacteria to form biofilm communities favouring theincorporation of their host into the biofilm (Ghigo, 2001). This wasdemonstrated for various bacteria, including some pathogens, butnever for Delftia.

B. Juarez Jimenez et al. / Journal of Biotechnology 157 (2012) 148– 153 151

Fig. 1. Development of Delftia tsuruhatensis BM90 on macroporous chemically modified cellulose BR-L01T70 as revealed by SEM observations at different incubation times.

Fig. 2. Development of Delftia tsuruhatensis BM90 on macroporous chemically modified cellulose BR-L01T70 as revealed by SEM observations (details). (A) Cells appearedto be attached to the carrier with fimbria-like structures, amorphous material seemed to be present and attached cells were in active division; (B) attached cells connectedby pili; (C) cells appeared to be attached to the carrier with fimbria-like structures; (D) intense colonization of carrier; arrows indicate cell aggregates (colonies); (E) detailof cell aggregate development on the carrier (arrow); and (F) cells appeared to be attached to the carrier with fimbria-like structures and some flagella seemed still present(arrow).

152 B. Juarez Jimenez et al. / Journal of Biotechnology 157 (2012) 148– 153

Fig. 3. Preliminary repeated batch process of Delftia tsuruhatensis BM90 immobi-lized on macroporous chemically modified cellulose BR-L01T70 and cultivated onRc

nto(otB

piald

3

aneb(dtaawBwivba

was minimal.

TY

Atbd

M for 312 h. Continuous line (�), immobilized cells; dashed line (�), free cells;ontinuous line (♦), control C1; dashed line (♦), control C2.

Even if production of exopolysaccharide (EPS) by our strain wasever recorded, the adhesion mechanism of BM90 could involvehe production of amorphous material (Fig. 2A). Carrier surface col-nization was sometime very intense; protuberant cell aggregatescolonies), grouped and surrounded by connecting material werebserved (Fig. 2D and E). These structures could be compared tohose described by the “water-channel-model” biofilm typology ofeer and Stoodley (1995).

Delftia are motile bacteria with scattered peritrichous orolar/bipolar flagella (Shigematsu et al., 2003; Han et al., 2005),

n our case it seems that very few cells maintained these structuresfter immobilization (Fig. 2F). It was not possible to understand ifoss of flagella was due to sample preparation artefacts or to theifferent cell physiological state caused by the immobilization.

.3. Repeated-batch processes in shaken culture

To understand the time course of phenols degradation by freend immobilized cells and to optimize batch duration, a prelimi-ary repeated batch process was carried out varying the length ofach batch in function of maximum degradation (Fig. 3). The firstatch (activation batch), necessary for cell activation and inductionFenice et al., 1998), was continued till the 96th h. However, degra-ation was faster during the first 48 h and other 24 h were necessaryo obtain further slight phenols removal to reach a plateau there-fter. This was recorded, even with slight differences, for both freend immobilized cells. Therefore, the subsequent batches durationas set to 72 h. It was evident that immobilized D. tsuruhatensisM90 was able to obtain maximum degradation after only 24 hhile the free cells needed double time (48 h). As for the controls,

t was evident that phenols adsorption to the carrier (ca. 2%) was

ery scarce and occurred mainly in the first 72 h of the activationatch to become almost negligible thereafter (C1). In addition, nobiotic phenols degradation was observed (C2).

able 3ield parameters (average) of the repeated batch process carried out with immobilized and

Averagebiomass (g L−1)

Specificdegradation (g g−1)

Immobilized 0.57a 0.79a

Free 0.80b 0.50b

verage biomass, average between biomass measured at the beginning and at the end of thhe average % of phenol degradation (90 and 80% for the immobilized and free cells, respecatch); specific productivity, g of phenol degraded per g of biomass per h. Column meansetermined by the Tukey test. SD was ca. 5%

Fig. 4. Optimized repeated batch process of Delftia tsuruhatensis BM90 immobilizedon macroporous chemically modified cellulose BR-L01T70 and cultivated on RM for360 h. Continuous line (�), immobilized cells; dashed line (�), free cells.

Fig. 4 reports the comparison between repeated batch carriedout using both free and immobilized BM90 cells under the opti-mized conditions. After the first activation batch (72 h), the processwas continued till the 360th h removing the exhausted mediumevery 24 and 48 h for immobilized and free cells, respectively. Theprocess was very stable and degradation rate was similar for eachbatch (ca. 90 and 80% for immobilized and free cells, respectively)and maintained for the whole process. Thus, immobilized BM90was definitely much more efficient, showing double productivitythan free cells, being able to degrade in the same time more thandouble amount of phenols. In other words, with the immobilizedcells double number of batches was possible in the same time span(12 and 6 batches for the immobilized and free cells, respectively,plus the activation batch, in 360 h).

In a previous work, phenol degradation of D. tsuruhaten-sis BM90 was recorded mainly during its exponential growth(Juarez-Jimenez et al., 2010). Very likely during the repeatedbatch process it was performed in its stationary phase. In fact,the attached biomass, measured at the end of the whole process(0.59 ± 0.06 g L−1), was not statistically different from that recordedafter the immobilization procedure (0.54 ± 0.07 g L−1). Same situa-tion was for recorded for the free cells even if with higher biomassproduction (0.78 ± 0.06 and 0.81 ± 0.08 g L−1, at the beginning andthe end of the process, respectively).

Table 3 resumes main repeated batch process yield parameters.High productivity apart, since the immobilized biomass was lowerthan that of free cells, also specific degradation activity was higher.

Finally, controls confirmed that phenols adsorption on the car-rier was very limited (C1) and almost no phenols degradation wasrecorded in cell free medium (C2) (data not shown). In addition,very little amount of free biomass grew during each batch (ca.0.034 ± 0.009 g L−1) and probably its contribute to the degradation

The degradation pattern of the immobilized cells of BM90, asdetected by GC–MS, was very similar to that reported by Juarez-Jimenez et al. (2010) for free living cells cultivated in batch

free cells of Delftia tsuruhatensis BM90 calculated per litre of fermentation medium.

Degradationproductivity (g h−1)

Specific productivity(g g−1 h−1)

0.019a 0.033a

0.008b 0.001b

e process; specific degradation, g of phenols degraded per g of biomass consideringtively); degradation productivity, g of phenol degraded per h (referred to the single

followed by the same superscript letter were not significantly different (P < 001) as

of Biot

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4

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A

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R

A

A

B

B

C

C

C

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F

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G

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B. Juarez Jimenez et al. / Journal

onditions (data not shown). Thus, the immobilization processeems to have no negative effect on the bacterium degradationathways.

All these features are very important for bioprocesses based onmmobilized cell systems (Leenen et al., 1996; Quiros et al., 1996;enice et al., 1998).

. Conclusions

In this study we selected a MPC carrier, BR-L01T70, for themmobilization of Delftia tsuruhatensis BM90 and we demonstratedts reusability, as a long-term biocatalyst for the biodegradation ofhenolic compounds, in a 360 h repeated-batch process. The immo-ilized bacterium showed high metabolic stability and improvedegradation ability (90% of phenols removal in 24 h for each batch)

f compared with the free cells. Therefore, it could be proposed, athe industrial level, as an efficient degrading system for phenolsontaining effluents.

cknowledgements

This work was done in the frame of the “Azioni integrate Italia-pagna 2006, Ministero dell’Università e della Ricerca Scientifica”nd “Acción Integrada Espana-Italia 2006, Ministerio Educacion yiencia”.

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