Effects of Mn 2+ and NH 4 concentrations on laccase and manganese peroxidase production and Amaranth decoloration by Trametes versicolor

Transcription

1 Appl Microbiol Biotechnol (1999) 51: 391±396 Ó Springer-Verlag 1999 ORIGINAL PAPER J. Swamy á J. A. Ramsay Effects of Mn 2+ and NH 4 concentrations on laccase and manganese peroxidase production and Amaranth decoloration by Trametes versicolor Received: 19 May 1998 / Received revision: 22 October 1998 / Accepted: 7 November 1998 Abstract Extracellular lignin peroxidase (LiP) was not detected during decoloration of the azo dye, Amaranth, by Trametes versicolor. Approximately twice as much laccase and manganese peroxidase (MnP) was produced by decolorizing cultures compared to when no dye was added. At a low Mn 2+ concentration (3 lm, N-limited (1.2 mm NH 4 ) cultures decolorized eight successive additions of Amaranth with no visible sorption to the mycelial biomass. At higher Mn 2+ concentrations (200 lm), production of MnP increased and that of laccase decreased, but the rate or number of successive Amaranth decolorations was una ected. There was always a 6-h to 8-h lag prior to decoloration of the rst aliquot of Amaranth, regardless of MnP and laccase concentrations. Although nitrogen-rich (12 mm NH 4 ) cultures at an initial concentration of 200 lm Mn 2+ produced high laccase and MnP levels, only three additions of Amaranth were decolorized, and substantial mycelial sorption of the dye occurred. While the results did not preclude roles for MnP and laccase, extracellular MnP and laccase alone were insu cient for decoloration. The cell-free supernatant did not decolorize Amaranth, but the mycelial biomass separated from the whole broth and resuspended in fresh medium did. This indicates the involvement of a mycelial-bound, lignolytic enzyme or a H 2 O 2 -generating mechanism in the cell wall. Nitrogen limitation was required for the expression of this activity. Introduction Research into dye decoloration by white rot fungi has largely focused on Phanerochaete chrysosporium and is J. Swamy á J. A. Ramsay (&) Department of Chemical Engineering, Queen's University, Kingston, Ontario, Canada K7L 3N6 ramsayj@chee.queensu.ca Tel.: Fax: attributed to lignin peroxidase (LiP) and manganese peroxidase (MnP), but not laccases. In the presence of H 2 O 2 and veratryl alcohol, P. chrysosporium LiP degrades several azo, triphenyl methane, heterocyclic and polymeric dyes (Cripps et al. 1990; Glenn and Gold 1983; Ollika et al. 1993; Pasti-Grigsby et al. 1992). Crude, cell-free extracts from ligninolytic cultures (Cripps et al. 1990; Ollika et al. 1993) and puri ed MnP (Pasti-Grigsby et al. 1992) from P. chrysosporium degrade certain dyes. Nitrogen (N) limitation induces LiP and MnP activity during decoloration by P. chrysosporium (Glenn and Gold 1983; Spadaro et al. 1992). There are no similar studies on dye decoloration by Trametes versicolor. N-limited ligninolytic cultures of T. versicolor have been shown to degrade PCBs, anthracene, uorene, phenanthrene, benzo[a]pyrene and dichloroaniline (Collins and Dobson 1996; Field et al. 1992; Morgan et al. 1991). However, N-rich cultures of T. versicolor decolorize kraft bleach plant e uent (BPE) and bleach kraft pulp (Addleman and Archibald 1993; Addleman et al. 1995; Archibald 1992; Roy and Archibald 1993) at high N concentrations that repress BPE decoloration by P. chrysosporium (Michel et al. 1991). High levels of extracellular laccase and MnP were detected under these conditions, and shown to play key roles in BPE decoloration by T. versicolor (Addleman et al. 1995; Paice et al. 1993). In previous work (J. Swamy and J. A. Ramsay, manuscript in preparation), decoloration by batch cultures of T. versicolor of repeated additions of di erent dyes and dye mixtures required adequate supplies of the primary carbon source, glucose, but was repressed in N- rich medium. The objectives of this study were to: (1) investigate the production of LiP, MnP and laccase by T. versicolor during sequential dye decoloration, (2) investigate the e ects of these enzyme concentrations on decoloration, (3) determine whether N limitation during dye decoloration is related to the expression of these enzymes, and (4) assess whether decoloration is caused by the activity of the extracellular enzymes.

2 392 Materials and methods Culture maintenance and medium T. versicolor ATCC was maintained on 2% (w/v) malt agar plates at 4 C. All studies were performed in autoclaved Kirk's medium (Kirk et al. 1978) at a ph of 5.0, with an initial glucose concentration of 10 g/l (56 mm). Ammonium tartrate was used as the nitrogen source. Mn 2+ and NH 4 concentrations were modi ed as described below. Preparation of inoculum T. versicolor from a malt agar plate was used to inoculate 200 ml of Kirk's medium in 500-ml Erlenmeyer asks. Four-day-old pellets incubated at 30 C and 200 rpm were used as inoculum. Sampling Samples (4 ml) were taken every 24 h from each ask and replaced with 4 ml of Kirk's medium without glucose or nitrogen. Samples were centrifuged to remove suspended biomass, and the concentrations of dye, glucose, NH 4, LiP, MnP and laccase in the supernatant were determined. All experiments in this study were performed in duplicate and the results are average values from triplicate sample analysis. E ect of Mn 2+ concentration on enzyme production and sequential Amaranth decoloration N-Rich (12 mm ammonium tartrate) and N-limited (1.2 mm ammonium tartrate) Kirk's medium with 3 lm and 200 lm Mn 2+ were prepared by adding the appropriate amounts of MnSO 4 á H 2 O. Then 200 ml of medium, in 500-ml Erlenmeyer asks, was inoculated with 30-ml portions of inoculum and incubated at 30 C and 200 rpm. The rst aliquot of 50 ppm Amaranth was added to all asks on day 0. After each complete decoloration, a subsequent 50 ppm Amaranth aliquot was added. A set of N-limited and N- rich cultures with 200 lm Mn 2+, to which no Amaranth was added, was maintained to determine enzyme production in the absence of dye. Decoloration of the rst Amaranth aliquot added at di erent times Two hundred millilitres of N-limited medium with 3 lm Mn 2+,in 500-ml Erlenmeyer asks, was inoculated with 30-ml portions of inoculum. The rst aliquot of 50 ppm Amaranth was added to the cultures on day 4, 9, 12 or 14. Following complete decoloration of the rst aliquot, two more 50-ppm aliquots were added to each ask. Decoloration by the whole broth, cell-free supernatant and resuspended biomass Two hundred millilitres of N-limited medium with 3 lm Mn 2+,in 500-ml Erlenmeyer asks was inoculated with 30 ml of inoculum. On day 0, 50 ppm Amaranth was added to all asks. Following complete decoloration of the rst aliquot, another was added on day 7. On day 9, the contents of one set of asks were centrifuged to separate the medium from the biomass, which was resuspended in 200 ml of fresh N-limited Kirk's medium with 3 lm Mn 2+. The supernatant was ltered through a sterile 21-lm-pore lter. Then 50 ppm Amaranth was added to the whole-broth 9-day-old culture, the resuspended biomass and the cell-fee supernatant and incubated at 30 C and 200 rpm. Analysis Amaranth was quanti ed spectrophotometrically at 523 nm using a linear standard curve. Glucose, NH 4, LiP, MnP and laccase concentrations were analysed in samples in which Amaranth was completely decolorized as it interferes with quanti cation. Glucose was determined using the 3,5-dinitrosalicylic acid test (Miller 1959); NH 4, by the phenol hypochlorite reaction (Weatherburn 1967). Laccase concentrations were measured by monitoring the oxidation of 0.27% (wt/vol) 2,2 -azinobis(3-ethylbenzothiazoline-6- sulfonic acid) (ABTS) at 420 nm (Archibald 1992; Wolfenden and Willson 1982). One unit of activity equals 1 lm ABTS oxidized per minute at 25 C and ph 5.0. LiP activity was measured by monitoring the oxidation of 2 mm veratryl alcohol to veratraldehyde at 310 nm with 0.1 mm H 2 O 2 in 100 mm tartrate bu er (ph 3.0) (molar extinction coe cient, 9300 M )1 cm )1 ) (Johansson and Nyman 1987). One unit of activity equals 1 lm veratryl alcohol oxidized per minute at 25 C. MnP activity was measured by monitoring change in optical density caused by formation of the malonate±mn 3+ complex at 270 nm with 50 mm Na malonate (ph 4.5), 0.2 mm MnSO 4 and 0.1 mm H 2 O 2 (Addleman et al. 1995; Gold et al. 1991). Amaranth, ABTS, 2,2 -dimethyl succinic acid, veratryl alcohol, Na malonate, H 2 O 2 and CaCl 2 were purchased from Aldrich. All other chemicals were obtained from Anachemia. Results Induction of decoloration A lag phase of approximately 6±8 h was observed during the decoloration of the rst addition of Amaranth, regardless of when it was added (Table 1). The rst aliquot was decolorized at rates of 1.2 to 1.5 ppm/h, with complete decoloration in about 48 h. Subsequent aliquots were decolorized more quickly (8.3±9.0 ppm/h), Table 1 Decoloration of the rst aliquot of Amaranth on day 4, 9, 12 or 14 by nitrogen-limited batch cultures of Trametes versicolor at 200 lm Mn 2+ (n.d. Not detected, MnP manganese peroxidase) Day First Amaranth addition Subsequent Amaranth addition Lag (h) Overall rate of decoloration (ppm/h) MnP Laccase No. of previous decolorations Lag (h) Overall rate of decoloration (ppm/h) MnP Laccase n.d. 18 ± ± ± n.d

3 393 with complete decoloration in shorter times of about 6h. E ect of Mn 2+ on N-limited cultures At initial concentrations of 56 mm glucose, 1.2 mm NH and 3 lm Mn 2+, glucose was consumed at a steady rate and exhausted by day 16, and NH 4 by day 6 (Fig. 1). The rst Amaranth aliquot was added on day 0; decoloration began on day 2 and was completed by day 4. No LiP was detected. Extracellular production of laccase and MnP began by day 2, with maximum levels of 1400 U/l laccase on day 12 and 60 U/l MnP on day 16 (Table 2). At a higher initial Mn 2+ concentration of 200 lm, glucose and NH 4 were consumed at a slightly higher rate and were exhausted earlier than previously (Fig. 2). However, the decoloration rate of the rst aliquot of dye was the same. No LiP was detected, but laccase and MnP production also began at day 2 with more MnP produced (Table 2). However, the maximum level of laccase was less. Despite signi cant di erences in enzyme production, cultures at both Mn 2+ concentrations rapidly decolorized eight successive additions of Amaranth, with no di erence in the extent or rate of decoloration (Table 2). Decoloration rates decreased and eventually ceased upon glucose depletion in both cases, despite su cient levels of extracellular laccase and MnP (Figs. 1, 2). Enzyme production in the absence of dye In the absence of dye, enzyme production by N-limited batch cultures at initial concentrations of 56 mm glucose, 1.2 mm NH 4 and 200 lm Mn 2+ was approximately half that achieved by decolorizing cultures, with maximum levels of 470 U/l MnP and 420 U/l laccase on day 12. In cultures with the same initial concentrations of glucose and Mn 2+ but 10 times more NH 4, glucose was exhausted relatively later (Fig. 3). The NH 4 concentration at this time was 5.59 mm. Enzyme production was again detected by day 2, and achieved relatively higher maximum levels of 1150 U/l MnP and 1810 U/l laccase on day 11 compared to cultures to which dye had been added. E ect of Mn 2+ on decoloration by N-rich batch cultures N-Rich cultures displayed limited ability for sequential decoloration (Table 2). Although there was no visible dye sorption to the mycelial biomass in N-limited cultures, there was substantial sorption of the third aliquot in N-rich cultures initially at 3 lm Mn 2+ and of the fourth aliquot in cultures initially at 200 lm Mn 2+. There was no further decoloration of the sorbed dye or the medium even after 7 days and glucose consumption ceased, suggesting inhibition of activity. Decoloration by cell-free supernatant and biomass Two sets of N-limited cultures, at an initial concentration of 3 lm Mn 2+, decolorized 50 ppm Amaranth added on days 4 and 7. On day 9, one set was centrifuged and ltered to separate the biomass from the supernatant. This cell-free supernatant did not decolorize Amaranth even when 0.1 or 1 mm H 2 O 2 was added. Addition of either amount of H 2 O 2 alone did not result in any decoloration. The separated biomass, resuspended in fresh medium, decolorized a third Amaranth aliquot within 24 h, and four subsequent additions, each within 6 h. The whole broth from the other set of cultures decolorized ve further aliquots, each within 6 h. Discussion Fig. 1 Substrate consumption and enzyme production during sequential decoloration by N-limited batch cultures at 3 lm Mn 2+. (MnP Manganese peroxidase) T. versicolor decolorized successive additions of di erent textile dyes and dye mixtures (J. Swamy, and J.A. Ramsay, manuscript in preparation), indicating its suitability for a decoloration process to treat textile ef- uent. Identi cation of the decoloration enzymes is useful for optimizing process parameters and medium composition, to maximize enzyme production and, therefore, decoloration. Under all experimental conditions in this study, extracellular MnP and laccase, but not LiP, were detected. Since decolorizing N-limited cultures produce approximately twice as much MnP and laccase as those to which no dye is added, these enzymes appear to be involved in decoloration. MnP and laccase are central to

4 394 Table 2 Laccase and MnP production by N-limited and N- rich batch cultures at 3 lm and 200 lm Mn 2+ with successive Amaranth additions 1.2 mm NH mm NH lm Mn lm Mn 2+ 3 lm Mn lm Mn 2+ Maximum MnP (U/L) Maximum laccase (U/L) Average rate of Amaranth decoloration (ppm/h) No. of decolorations T. versicolor BPE decoloration, and a MnP-de cient mutant has a greatly reduced ability to decolorize the polymeric dyes poly-b411 and poly-r478 compared to the parent strain (Addleman et al. 1995). Furthermore, puri ed P. chrysosporium MnP can oxidize a number of dyes (Pasti-Grigsby et al. 1992). MnP concentration correlates strongly with the extent of BPE decoloration by T. versicolor (Addleman and Archibald 1993). Augmenting the Mn 2+ concentration in N-limited cultures from 3 lm to 200 lm increased MnP production and decreased laccase production. However, in contrast to BPE decoloration, the rate and number of Amaranth decolorations were una ected. The expression of the degradative enzymes by most white rot fungi requires N limitation (Field et al. 1992; Reddy 1995). In this work, N limitation was required to sustain dye decoloration by T. versicolor. However, N- rich cultures produced higher levels of MnP and laccase than N-limited cultures. This clearly indicates that inhibited decoloration at high N concentrations is not due to the absence of either of these enzymes. While the results do not preclude the involvement of extracellular MnP or laccase, several factors indicate that these enzymes alone are insu cient for decoloration. Decoloration by N-limited cultures ceased with Fig. 3 Substrate consumption and enzyme production by N-rich batch cultures at 200 lm Mn 2+ glucose depletion, despite high extracellular concentrations of MnP and laccase. N-rich cultures and the cell-free supernatant from N-limited cultures cannot decolorize dye, even though both contain high MnP and Fig. 2 Substrate consumption and enzyme production during sequential decoloration by N-limited batch cultures at 200 lm Mn 2+ Fig. 4 Amaranth decoloration by a 9-day-old culture (whole broth), resuspended biomass and cell-free supernatant

5 395 laccase concentrations. Furthermore, the rst addition of Amaranth is always decolorized at a lower rate, with a lag period of 6±8 h, regardless of the MnP and laccase concentrations at the time of dye addition. Subsequent aliquots were decolorized more rapidly. This implies that dye addition induces essential decoloration enzyme(s) or factor(s) other than MnP and laccase. Decoloration requires a biomass-associated component since: (1) there was no decoloration by the cell-free supernatant despite high concentrations of laccase and MnP, and (2) there were high rates of decoloration by the resuspended biomass. Substantial amounts of dye were sorbed to the biomass in N-rich cultures and were not further decolorized, even though there was no visible sorption in N-limited cultures. A preliminary step in the decoloration mechanism may involve the dye binding to the mycelium, perhaps for initial degradation by a biomass-associated component, the expression and/or activity of which is induced when N is limited so that, under this condition, the bound dye is decolorized too rapidly for visible sorption to be observed. Kirk and Farrell (1987) have demonstrated an example in which lignin must bind to the fungal wall to be degraded. A possible biomass-associated product is H 2 O 2, a cosubstrate for oxidation by ligninolytic peroxidases (Daniel et al. 1994; Kelly and Reddy 1988; Kersten and Kirk 1987; Volc and Eriksson 1988), or a lignolytic enzyme. Glucose-2-oxidase (pyranose oxidase) is a constitutive peroxidase in T. versicolor found between the cell membrane and cell wall. This is believed to be the site of H 2 O 2 production (Daniel et al. 1994; Machida and Nankanishi 1984). D-Glucose is its preferred substrate and the carbon source used in this study. Decoloration was shown to require glucose and ceased with glucose depletion. Glucose-2-oxidase may be a major source of H 2 O 2 and may partially explain the requirement for both glucose and the biomass for decoloration. Immunochemical methods have revealed that a fraction of the LiP produced by P. chrysosporium remains associated with the fungal wall (Garcia et al. 1987) and washed pellets have been shown to retain partial lignindegrading ability (Kurek and Odier 1990). Acknowledgements The authors acknowledge the Natural Science and Engineering Council of Canada for support of this work. J. Swamy is a recipient of a Queen's University Graduate Award. References Addleman K, Archibald F (1993) Kraft pulp bleaching and deligni cation by dikaryons and monokaryons of Trametes versicolor. 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