Appl Microbiol Biotechnol (1998) 49: 584±588
Ó Springer-Verlag 1998
ORIGINAL PAPER
M. Hofrichter á K. Scheibner á I. Schneegaû D. Ziegenhagen á W. Fritsche
Mineralization of synthetic humic substances by manganese peroxidase from the white-rot fungus Nematoloma frowardii
Received: 25 September 1997 / Received revision: 12 January 1998 / Accepted: 13 January 1998
Abstract A manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii was found to be capable of releasing up to 17% 14CO2 from 14C-labelled synthetic humic substances. The latter were prepared from [U-14C]catechol by spontaneous oxidative polymerization or laccase-catalysed polymerization. The extent of humic substance mineralization was considerably enhanced in the presence of the thiol mediator glutathione (up to 50%). Besides the evolution of 14CO2, the treatment of humic substances with Mn peroxidase resulted in the formation of lower-molecular-mass products. Analysis of residual radioactivity by gel-permeation chromatography demonstrated that the predominant molecular masses of the initial humic substances ranged between 2 kDa and 6 kDa; after treatment with Mn peroxidase, they were reduced to 0.5±2 kDa. The extracellular depolymerization and mineralization of humic substances by the Mn peroxidase system may play an important role in humus turnover of habitats that are rich in basidiomycetous fungi.
Introduction Humic substances are organic materials built up in soils as well as in waters and are also present in low-rank coals (lignites). They derive from postmortal materials and are formed by numerous reactions (Ziechmann 1994). The theory is that humic substances represent modi®ed lignins which additionally contain amino compounds and carbohydrates (Stevenson 1994). The genesis of humic substances has been well investigated using the auM. Hofrichter (&) á K. Scheibner á I. Schneegaû D. Ziegenhagen á W. Fritsche Friedrich-Schiller-UniversitaÈt Jena, Institut fuÈr Mikrobiologie, Lehrstuhl Technische Mikrobiologie, Philosophenweg 12, D-07743 Jena, Germany Tel.: +49 3641 630950 Fax: +49 3641 631237 e-mail:
[email protected]
toxidation of phenols as a model reaction for humi®cation (Eller 1921; Ziechmann 1994). In contrast, less is known about the degradation of humic substances. The ability to depolymerize and mineralize soil humic acids has been described for streptomycetes (Kontchou and Blondeau 1992) and basidiomycetes (Burges and Latter 1960; Blondeau 1989). Activities of lignin peroxidase and manganese (Mn) peroxidase were determined in cultures of the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor, which grew in the presence of natural or synthetic humic substances (Dehorter and Blondeau 1992). Furthermore, there are a few reports on the degradation of coal-derived humic substances by basidiomycetous fungi (Ralph and Catcheside 1994; Willmann and Fakoussa 1997). We reported previously that the white-rot fungi Nematoloma frowardii and Clitocybula dusenii are capable of eectively depolymerizing coal-derived humic substances in solid agar media (Hofrichter and Fritsche 1996, 1997a). Moreover, Mn peroxidase was found to be the crucial enzyme in the degradation process, which was proven by the depolymerization of coal humic substances in a cell-free system (Hofrichter and Fritsche 1997b; Ziegenhagen et al. 1997). The present paper demonstrates that the depolymerization of synthetic humic substances by a Mn peroxidase preparation from N. frowardii is accompanied by the release of carbon dioxide.
Materials and methods Organism The South American white-rot fungus Nematoloma frowardii (Horak) b19 (DSM 11239, ATCC 201144) was isolated and characterized as described previously (Hofrichter and Fritsche 1996, 1997a). Culture conditions and Mn peroxidase preparation A nitrogen-sucient medium containing 300 lM MnCl2 was used for the production of Mn peroxidase (Hofrichter and Fritsche 1997b). Conical ¯asks containing 300 ml medium were inoculata-
585 ted with ten agar plugs obtained from 14-day-old malt-extract agar plates. The surface cultures were harvested after 25 days of cultivation and ®ltered through glass wool. The Mn peroxidase containing ®ltrate was concentrated and dia®ltered as described recently (Schneegaû et al. 1997). Crude laccase from N. frowardii was produced using the same medium and conditions as for Mn peroxidase production (Hofrichter and Fritsche 1997b), except that MnCl2 was replaced by 2,5xylidine (165 lM) as elicitor. Cultures were harvested after 6 days of surface cultivation, and were concentrated and dia®ltered (10 mM citrate buer, pH 5.0) by two steps of ultra®ltration (Ultralab 2L, Minisette tangential ®lter Omega 10-kDa molecular mass cut-o; Pall Filtron, Karlstein, Germany; Amicon chamber, radial ®lter, polysulphonate, 10-kDa cut-o). The ®nal laccase preparation contained an activity of 20.8 U ml)1; activities of the peroxidases could not be determined. Preparation of synthetic
14
C-labelled humic acids
14
Synthetic U- C-labelled humic acids were prepared from [U-14C]catechol (speci®c radioactivity 2.0 mCi mmol)1 74 MBq mmol)1; Sigma, St. Louis, Mo.) by spontaneous oxidative polymerization (P¯ug 1980; Ziechmann 1994) or by laccase-catalysed polymerization (Bollag et al. 1988). Spontaneous oxidative polymerization [14C]Catechol (462.5 kBq 2.75 ´ 107 dpm) and 9.4 lmol unlabelled catechol were dissolved in 1 ml 0.1 M NaOH also containing Fe3+ and Cu2+ (®nal concentration 10 mM) to enhance the oxidative polymerization (Ziechmann 1994). The reaction mixture was incubated on a rotary shaker (200 rpm) at 37 °C. After 7 days, the humic substances (HS) were precipitated by addition of 50 ll HCl (36%, resulting pH
