Treatment of effluents containing phenols such as textile dyes with fungal laccases is usually limited to the acid to neutral pH range and moderate temperatures. Here we demonstrate for the first time that spore-bound laccases which are stable at high temperatures and pH values can be used for phenolic dye decolorisation. Laccase containing spores from Bacillus SF were immobilized on alumina pellets. Both immobilized and free spores were able to
completely decolorize the common textile dyes Mordant Black 9, Mordant Brown 96/Mordant Brown 15, and Acid Blue 74 within 90 min of incubation time and decolorized solutions were successfully used in re-dyeing.
spores from Bacillus SF were immobilized on alumina pellets. Both immobilized and free spores were able to completely decolorize the common textile dyes Mordant Black 9, Mordant Brown 96/Mordant Brown 15, and Acid Blue 74 within 90 min of incubation time and decolorized solutions were successfully used in re-dyeing.European
Project BIOEFFTEXCompetence Centre Applied Biocatalysi

Gübitz, Georg M.

Held, C.

Kandelbauer, A.

Paulo, Artur Cavaco

Schroeder, M.

Environmental Chemistry Letters

Treatment of effluents containing phenols such as textile dyes with fungal laccases is usually limited to the acid to neutral pH range and moderate temperatures. Here we demonstrate for the first time that spore-bound laccases which are stable at high temperatures and pH values can be used for phenolic dye decolorisation. Laccase containing spores from Bacillus SF were immobilized on alumina pellets. Both immobilized and free spores were able to
completely decolorize the common textile dyes Mordant Black 9, Mordant Brown 96/Mordant Brown 15, and Acid Blue 74 within 90 min of incubation time and decolorized solutions were successfully used in re-dyeing.
spores from Bacillus SF were immobilized on alumina pellets. Both immobilized and free spores were able to completely decolorize the common textile dyes Mordant Black 9, Mordant Brown 96/Mordant Brown 15, and Acid Blue 74 within 90 min of incubation time and decolorized solutions were successfully used in re-dyeing.This study was supported by the European Project BIOEFFTEX and the Competence Centre Applied Biocatalysi

Universidade do Minho: RepositoriUM

Environ Chem Lett (2005) 3: 74–77DOI 10.1007/s10311-005-0006-1ORIGINAL PAPERC. Held · A. Kandelbauer · M. Schroeder ·A. Cavaco-Paulo · G. M. GuebitzBiotransformation of phenolics with laccase containingbacterial sporesReceived: 5 May 2005 / Accepted: 9 August 2005 / Published online: 22 October 2005C© Springer-Verlag 2005Abstract Treatment of effluents containing phenols suchas textile dyes with fungal laccases is usually limited to theacid to neutral pH range and moderate temperatures. Herewe demonstrate for the first time that spore-bound laccaseswhich are stable at high temperatures and pH values canbe used for phenolic dye decolorisation. Laccase contain-ing spores from Bacillus SF were immobilized on aluminapellets. Both immobilized and free spores were able tocompletely decolorize the common textile dyes MordantBlack 9, Mordant Brown 96/Mordant Brown 15, and AcidBlue 74 within 90 min of incubation time and decolorizedsolutions were successfully used in re-dyeing.Keywords Bacillus . Spores . Biodegradation ofphenolics . Effluent treatmentIntroductionLaccases (benzenediol:oxygen oxidoreductase, EC1.10.3.2) have found several applications in enzyme re-mediation. Laccases as well as immobilized laccases havebeen used for the treatment of phenolic effluents, PAHs andPCBs. The enzymes render phenolic compounds less toxicvia degradation or polymerization reactions and/or cross-coupling of pollutant phenols with naturally occurringphenols (Bohmer et al. 1998; D’Annibale et al. 2000; Ullahet al. 2000). Besides microbial processes (Fischer-Colbrieet al. 2005), laccases have also a potential for the decol-orization of textile dyes (Kandelbauer and Guebitz 2005).C. Held · A. Kandelbauer · M. Schroeder · G. M. Guebitz ()Department of Environmental Biotechnology, Graz Universityof Technology,Petersgasse 12,8010 Graz, Austriae-mail: guebitz@tugraz.atTel.: +43-316-873-8312Fax: +43-316-873-8815A. Cavaco-PauloDepartment of Textile Engineering, University of Minho,4800 Guimaraes, BrazilLaccases catalyze the removal of a hydrogen atom fromthe hydroxyl group of ortho and para-substituted mono-phenolic and poly-phenolic substrates and from aromaticamines by one-electron abstraction, to form free radicals,capable of undergoing further depolymerization, repoly-merization, demethylation or quinone formation.For dye decolorization, the laccases from a number offungi such as Sclerotium rolfsii, Trametes modesta, T. hir-suta, T. versicolor, Pleurotus pulmonarius among othershave been investigated (Kandelbauer and Guebitz 2005).However, the potential of bacterial laccases for this pur-pose has not been assessed so far. In contrast to fungallaccases which are known for many years, laccase activityin bacteria has only been described in 1993 for Azospir-illum lipoferum and has been characterized later in moredetail (Diamantidis et al. 2000). Interestingly, the spore coatprotein CotA of Bacillus subtilis was identified as laccase(Hullo et al. 2001). Since spores serve microorganisms tosurvive drastic conditions, spore coat enzymes might alsowithstand high temperatures or extreme pH values whichwould be advantageous for industrial applications.It has previously been shown that residual enzyme afterenzymatic decolorization of dyeing effluents can restrictthe reuse of water (Abadulla et al. 2000). Enzyme proteincan adsorb to fabrics and thus lead to unwanted color differ-ences in dyeing with enzymatically decolorized effluents.Therefore, for continuous applications in dye decoloriza-tion and also in other applications immobilization of lac-cases is required and several successful examples have beenreported (Abadulla et al. 2000; Kandelbauer et al. 2004).In a previous study higher decolorization rates were re-ported with an immobilized laccase from the white-rot fun-gus Trametes modesta when compared to the free enzyme.Despite the promising efficiency of dye decolorization ingeneral, no decolorization activity at all was observed atpH values higher than pH 7 (Kandelbauer et al. 2004). Thislimits the industrial potential since many dyeing processes(e.g., cotton) are performed in the alkaline pH range. Alter-natively, spore laccases which are also active in the alka-line pH range could be used for dye decolorization withoutliberation from spores allowing simple immobilization of75spores or application in membrane reactors. In this study,we report for the first time the use of a spore laccase fromBacillus SF for dye decolorization.ExperimentalCultivation of Bacillus SFBacillus SF was isolated as described previously (Gudeljet al. 2001). Cultures were grown on Caso-agar (Merck)plates pH adjusted with NaOH to 8.5 for 2 days at 55◦C andthereafter stored at 4◦C. The medium for liquid cultivationof Bacillus SF consisted of two different solutions whichwere combined after autoclaving. Solution A (g l−1): 3.5KH2PO4, 4.98 NaHPO4·2H2O, 2.5 (NH4)2SO4, 10 yeastextract, 10 peptone from casein l, 60 ml 1 M NaOH,Solution B (g l−1): 4.5 MgSO4·7 H2O, 0.2 MnSO4·H2Ol−1,100 ml iron citrate (8,9 g l−1), 25 ml trace element so-lution SL6 l−1, 10 ml 1 M NaOH l−1. The medium for theinduction of sporulation of Bacillus SF consisted of (g l−1)8 nutrient broth (Merck), 1 KCl, 0.25 MgSO4·7H2O, andafter autoclaving (121◦C, 30 min) sterile 1 ml l−1 of the fol-lowing solutions was added 1 M Ca(NO3)2·4H2O, 0.01 MMnCL2, 0.001 M FeSO4, and 0.5 ml of 1 M NaOH solu-tion. The 1 l baffled Erlenmeyer flasks containing 250 mlof the above medium were inoculated with Bacillus SF ac-tively growing on agar plates and incubated at 55◦C on arotary shaker (130 rpm) for 3 days and then 5 ml l−1 weretransferred to the sporulation medium.Immobilization of the sporesAlumina pellets (Sigma) were silanized twice at 50◦C for24 h in a 3% (v/v) solution of aminopropyl triethoxy silane(Sigma) in acetone. The silanized pellets were washed withdistilled water and immersed in 2.5% (v/v) aqueous glu-tardialdehyde solution (Sigma) for 2 h at 20◦C. Thereafter,the pellets were incubated with 60 mg spores l−1 for 5 hat 20◦C. The immobilized spore pellets were washed withbuffer (pH 8.5) and kept refrigerated until further use. Ef-fective laccase activity of solution was determined beforeand after immobilization.Laccase activityBefore its usage in decolorization, the effective activities ofthe laccase and laccase activity of spore dispersions weredetermined using the oxidation of 2,6-dimethoxyphenol(DMP). The reaction mixture contained 50 mM sodiummalonate (pH 4.5), 1 mM DMP, 1 mM MnSO4, and 700 µlsample in a total of 1 ml. The formation of 2,2′,6,6′-dimethoxydiphenoquinone at 30◦C was followed spec-trophotometrically at 468 nm and apparent laccase ac-tivity was calculated from the molar extinction coefficientε=49.6 mM−1 cm−1.Isolation of the laccase from the sporesCulture (40 ml) were harvested through centrifugation at9000×g for 30 min. The supernatant was withdrawn andthe pellet was washed twice through resuspension in 10 mlof deionized water and centrifugation (9000×g, 15 min).After a final centrifugation at 16000×g for 20 min cells(spores) were resuspended in an equal volume of a solu-tion containing 0.1 M NaOH, 8.0 M urea, 1% sodium-dodecylsulfate. Cells were incubated for 15 min at 25◦Cand subsequently centrifuged at 16000×g for 20 min. Thesupernatant was filtrated through 0.22 µm membrane fil-ter (Osmonics Inc., Germany) at 2 bar positive pressure.Sodiumdodecylsulfate (SDS) was removed from the crudeenzyme preparation through precipitation with an equalamount of 0.5 M potassium acetate buffer pH 5 as potas-sium dodecylsulfate (PDS). After incubation for 15 minat 25◦C PDS precipitate was removed through centrifuga-tion at 16000×g for 20 min and filtration through 0.22 µmmembrane filter. Urea was removed through centrifugationwith vivaspin (Satorius AG, Germany) centrifugal concen-trators molecular cutoff 5 kDa and exchanging the bufferwith 0.2 M N,N,N-tris(hydroxymethyl) aminomethane hy-drochloride buffer pH 8.5.Temperature and pH-stabilitiesThe thermal stability of the laccase was examined by prein-cubation in 10 mM phosphate buffer pH 8.0 of free andimmobilized spores at various temperatures and measure-ment of remaining activity with the assay described above.The pH-stabilities were determined similarly incubatingfree and immobilized spores at 25◦C in different buffersranging from pH 5.0 to 9.5.Dye decolorization experimentsFor decolorization experiments, the commercial textiledyes Diamond Black PV 200 (CI Mordant Black 9 (DBPV200)), Diamond Fast Brown (CI Mordant Brown 96/CIMordant Brown 15 (DFB)) kindly donated from DyeStarand Indigo Carmine (IC) (Acros) were used. Aqueous so-lutions containing 50 mg l−1 dye treated with 5 g l−1 sporesor 7 g l−1 immobilized spores at 60◦C for 90 min. Sam-ples were withdrawn every 5 min and analyzed by UV/Visspectroscopy using a U-2001 Hitachi spectrophotometer inthe spectral range of 240–800 nm with a scan speed of1200 nm min−1. To account for the effects of temperatureand pH, controls were performed without spores while thetypical laccase inhibitor NaF (1 mM) was added to accountfor possible adsorption effects.Dyeing experimentsBleached cotton fabrics (10×10 cm) were dyed with ei-ther CI Reactive Orange 70 or CI Reactive Blue in liquorscontaining enzymatically decolorized dyes and 20 g l−176Na2CO3 and 60 g l−1 Na2SO4. An Ahiba Spectradye dye-ing apparatus (Datacolor International, Luzern, Switzer-land) was used at a liquor to good ratio of 20:1 (40 rpm,step 1: temperature was raised from 20 to 60◦C in 20 min;step 2: 60◦C, 60 min). Dye load was either low (0.5%) orhigh (3%) with respect to the dry mass of the fabric. Dyedfabrics were washed at the same liquor ratio with the non-ionic detergent Hostapal CV (1 g l−1) for 30 min at 90◦Cto remove the unfixed dye. Diode array spectrums (TIDASinstrument from J and M, Aalen, Germany) of dyes both instandard dye baths and in dye baths containing enzymati-cally decolorized dyes were recorded.Color differences of the dyed fabrics were determinedusing a reflectance measuring apparatus (Spectraflash 600from Datacolor, Luzern, Switzerland) according to theCIELAB color difference concept at standard illuminantD65 (LAV/Spec. Excl., d/8, D65/10◦) with a color toler-ance interval of 1 CIELAB unit. Color deviation was alsoquantified by calculating the K/S values from reflectancemeasurements on the dyed fabrics.Results and discussionLaccase activityThe activity of the Bacillus SF spore bound laccase wasdependent on the age of the spores. Maximum activitywas achieved after 9 days of growth. Thereafter, activityremained constant within the observed time of 14 days.Laccase activities of spores were 0.64 U mg−1 dry weightof spores. The effective laccase activity of the immobilizedspores was 0.48 U mg−1 (based on the amount of sporesimmobilized) indicating that immobilization partially in-activated the laccase and/or restricted the access of thesubstrate to the enzyme.Several procedures for the isolation of the laccase fromspores were compared (data not shown) and only a drastictreatment led to release of active laccase (6%). Most likelythis low activity recovery is due to incomplete re-naturationof the enzyme.Temperature stabilitiesThe temperature stability (half life times t1/2) of the freeBacillus SF laccase liberated from spores was only 5 minat 60◦C and pH 8.0. In contrast, the observed half life timesof laccase activity of Bacillus SF spores revealed high tem-perature stabilities of 66 h at 60◦C and pH 8.0. These valuesare about six and 20 times higher than stabilities of fungallaccases reported previously for this temperature and opti-mum pH values of the individual enzymes (11.5 h, Abadullaet al. 2000 and 3 h, D’Annibale et al. 2000). At 80◦C thehalf life was still 30 min. Immobilization of the Bacillus SFspores onto alumina pellets slightly increased the half lifetimes from 66 to 80 h at 60◦C. Similarly, increased temper-ature stabilities have been reported previously for fungallaccases. (Abadulla et al. 2000; D’Annibale et al. 2000).pH stabilitiesThe pH half life time of the free Bacillus SF laccase was48 h at pH 8.5. However, the laccase activity of Bacil-lus SF spores showed higher stabilities over a broad pHrange. Within a pH range from 5.0 to 7.0 the half lifewas more than 120 h compared to 13 h previously mea-sured for a laccase from Trametes hirsuta (Abadulla et al.2000). In contrast to fungal laccases, laccase activity ofBacillus SF spores showed a very high stability at alka-line pH values (t1/2=67 h at pH 9.5). These observed halflife times indicate a high potential for industrial processeslike dye decolorization, detoxification, and transformationof phenolic and other compounds where high temperaturesand pH values are common. Previously, an intracellularcatalase-peroxide from this Bacillus SF has been describedwhich also showed high stabilities at alkaline pH and hightemperatures (Gudelj et al. 2001).Dye decolorizationBased on the high stability of laccase activity of Bacillus SFspores compared to the liberated enzyme, only spores andimmobilized spores were investigated for dye decoloriza-tion. The spore laccases as well as the spores immobilizedon inorganic carrier material were used for the decoloriza-tion of the common textile dyes DBPV 200 (CI MordantBlack 9), DFB (CI Mordant Brown 96/CI Mordant Brown15) and IC (CI Acid Blue 74) in order to demonstratetheir potential for the treatment of process waters contami-nated with excess dyestuff. Both types of spore preparationsproved to be very efficient in the complete removal of colorwithin less than 90 min of incubation time.Decolorization rates depended upon dye structure. DBPV200, DFB, and IC were almost completely decolorized af-ter 35, 50, and 65 min, respectively (Fig. 1). A similar trendwas found earlier when an immobilized laccase from thewhite-rot fungus Trametes modesta was used to treat thesame dyes (Kandelbauer et al. 2004). Although the opti-mum decolorization rates were generally higher with theimmobilized fungal laccase, they were strongly dependenton acidic conditions and no decolorization activity at allwas observed at pH values higher than pH 7. In contrast,when using the bacterial spore preparations, decolorizationwas achieved at pH 9. Furthermore, in previous experimentswith fungal laccases, the optimum temperature for dye de-colorization was never above 40◦C whereas in the presentexperiments the best results were found at 60◦C. No sig-nificant decolorization was seen when the laccase inhibitorNaF was present in the incubation mixtures indicating thatadsorption did not contribute to decolorization.Our results indicate access of substrates to the outer sporecoat protein of Bacillus sp. displaying laccase activity doesnot seem to be sterically limited. This is in agreementwith previous data for immobilized fungal laccases. Con-sequently, in different general decolorization rates of struc-turally different dyes are rather due to presence of differentelectron withdrawing of electron donating substituents on770204060801000 20 40 60 80 100time [min]Residual dye [%]DBPV200DFBIndigo CarmineFig. 1 Decolorization of textile dyes with immobilized spore-boundlaccase from Bacillus SFthe aromatic rings than due to sterical effects (Kandelbaueret al. 2004). In the next section it will be shown that thespore-treated dye solutions can be re-used for dyeing oper-ations.Cotton dyeingRe-using the laccase-treated effluent in the dyeing processwe have found previously that enzyme protein obviouslystrongly interfered with the dyeing process while promis-ing results were obtained with the immobilized laccases(Abadulla et al. 2000). This was indicated by dE∗ values ofthe dyed fabrics below 1.1, which is acceptable to the indus-try. Shifts in dye absorption maximum in solution could becaused by binding of auxochromes to the dye molecules.Alternatively, degradation products and/or protein couldcause aggregation of dye molecules preventing the dye up-take to the fabric, which would cause larger color fail. Thus,immobilization of enzymes seems to be strictly required inorder to allow reuse of enzyme-treated effluents.Decolorized solutions of the three commercial dyesDBPV 200, DFB, and IC were used for the dyeing ofbleached cotton with reactive dyes. Results are shown inFig. 2. Even with low dye concentrations, no significantcolor differences between fabrics dyed in baths preparedwith water or enzyme-treated dye solutions were seen. Onlyin the case of enzyme-treated solutions of IC high dye con-centrations were necessary in a second dyeing step to reachacceptable dE∗ differences of the dyed fabrics below 1.1.ConclusionIn this study we have shown for the first time that sporesfrom Bacillus SF with laccase activity can be used forcontinuous decolorization of dyes when immobilized onalumina carriers. Despite some loss of laccase activity dueto the immobilization procedure both pH and temperaturestabilities of laccase activity of the immobilized sporeswere higher than those of the free spores. Dye solutionstreated with spore laccase can be used for the preparationof new dyeing baths leading to acceptable color differencesof around 1 dE.00,511,522,53Low High Low High Low HighDBPV200 DFB Indigo CarminedECI Reactive OrangeCI Reactive BlueFig. 2 Color difference (dE) between cotton fabrics dyed with Re-active Orange 70 or Reactive blue 214 (high and low concentration)either in water or decolorized dye solutions. Dye solutions contain-ing the textiles dyes DBPV 200, DFB, and IC were decolorized withimmobilized spore laccase from Bacillus SFAcknowledgements This study was supported by the EuropeanProject BIOEFFTEX and the Competence Centre Applied Biocatal-ysisReferencesAbadulla E, Tzanov T, Costa S, Robra KH, Cavaco-Paulo A, Gu¨bitzGM (2000) Decolorization and detoxification of textile dyeswith a laccase from Trametes hirsuta. Appl Environ Microbiol66:3357–3362Bohmer S, Messner K, Srebotnik E (1998) Oxidation of phenanthreneby a fungal laccase in the presence of 1-hydroxybenzotriazoleand unsaturated lipids. Biochem Biophs Res Commun244:233–238D’Annibale A, Stazi SR, Vinciguerra V, Sermanni GG (2000)Oxirane-immobilized Lentinula edodes laccase: stabilityand phenolics removal efficiency in olive mill wastewater. JBiotechnol 77:265–273Diamantidis G, Effosse A, Potier P, Bally R (2000) Purificationand characterization of the first bacterial laccase in the rhizo-spheric bacterium Azospirillum lipoferum. Soil Biol Biochem32:919–927Fischer-Colbrie G, Maier J, Robra K-H, Gu¨bitz GM (2005)Degradation of the indigo carmine dye by an anaerobic mixedpopulation. In: Lichtfouse E, Dudd S, Robert D (eds) Environ-mental chemistry. Springer-Verlag, Heidelberg, pp 289–294Gudelj M, Fruhwirth G, Paar A, Lottspeich F, Robra KH, Cavaco-Paulo A, Gu¨bitz GM (2001) A catalase-peroxidase from anewly isolated thermoalkalophilic Bacillus sp. with potentialfor the treatment of textile bleaching effluents. Extremophiles5:423–429Hullo MF, Moszer I, Danchin A, Martin-Verstraete I (2001) CotAof Bacillus subtilis is a copper-dependent laccase. J Bacteriol183:5426–5430Kandelbauer A, Guebitz GM (2005) Bioremediation for thedecolorization of textile dyes, a review. In: Lichtfouse E,Schwarzbauer J, Robert D (eds) Environmental chemistry.Springer-Verlag, Heidelberg, pp 269–288Kandelbauer A, Maute O, Kessler RW, Erlacher A, Gubitz GM(2004) Study of dye decolorization in an immobilized laccaseenzyme-reactor using online spectroscopy. Biotechnol Bioeng87:552–563Ullah MA, Bedford CT, Evans CS (2000) Reactions of pen-tachlorophenol with laccase from Coriolus versicolor. ApplMicrobiol Biotechnol 53:230–234

Biotransformation of phenolics with laccase containing bacterial spores

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Biotransformation of phenolics with laccase containing bacterial spores

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