Appl Microbiol Biotechnol (2000) 53: 441±446
Ó Springer-Verlag 2000
ORIGINAL PAPER
U. Wunderwald á G. Kreisel á M. Braun á M. Schulz C. JaÈger á M. Hofrichter
Formation and degradation of a synthetic humic acid derived from 3-¯uorocatechol
Received: 28 June 1999 / Received revision: 14 October 1999 / Accepted: 16 October 1999
Abstract A synthetic ¯uorinated humic acid (FHA) was prepared by the spontaneous oxidative polymerization of 3-¯uorocatechol. The 13C-solid-state NMR spectrum showed signals in the region for aromatic carbons with dierent substituents (aryl-H, aryl-C, aryl-O carbons) and for carboxyl-carbon. The latter indicated the formation of carboxylic groups, probably caused by ring cleavages during the polymerization process. An indication of the formation of carboxylic groups was also found in the infrared spectrum (band at 1715 cm)1). The dissolved FHA was degraded with active mycelium of the agaric white-rot fungus Nematoloma frowardii as well as with its isolated manganese peroxidase. In both cases, decolorization of the brownish FHA solution and partial de¯uorination (45±60%) took place. Degradation proceeded via formation of lower-molecular-mass fulvic acid-like substances. The results demonstrate that halogenated humic substances, e.g., resulting from the humi®cation of xenobiotic compounds (bound residues), can in principle be eliminated by ligninolytic fungi (e.g., soil colonizing litter decomposers) and their manganese peroxidase system.
U. Wunderwald á G. Kreisel Institut fuÈr Technische Chemie, Friedrich-Schiller-UniversitaÈt, Jena, Germany M. Braun á M. Schulz á C. JaÈger Institut fuÈr Optik und Quantenelektronik, Friedrich-Schiller-UniversitaÈt, Jena, Germany M. Hofrichter Lehrstuhl fuÈr Angewandte und OÈkologische Mikrobiologie, Friedrich-Schiller-UniversitaÈt, Jena, Germany U. Wunderwald (&) GSF-Forschungszentrum fuÈr Umwelt und Gesundheit GmbH, Institut fuÈr Biochemische P¯anzenpathologie, Geb. 57, IngolstaÈdter Landstrasse 1, 85764 Neuherberg, Germany e-mail:
[email protected] Tel.: +49-089-31873473 Fax: +49-089-31872726
Introduction Phenols are suitable compounds for the study of the genesis of humic acids (Ziechmann 1994). Because they both take part in electron donor acceptor complexes and form radicals, it is possible to obtain a series of humic acids made from dierent phenols. On the one hand, phenols themselves form humic acid-like compounds under alkaline solutions in the presence of O2 (spontaneous oxidative polymerization), and on the other, they can be introduced into existing humic substances, altering them partially. The latter occurs in nature when phenolic compounds (both natural and man-made) get into soil or water. The intensity of the change of humic acids depends on the kind of bonds formed between the humic acids and the phenols. As a result of covalent binding of phenols, the modi®ed humic acids show structural characteristics of the original phenols. Such modi®cations can aect microorganisms and plants as well as their enzymes interacting with humus, especially if halogenated phenols are incorporated (Ziechmann 1996). So far, investigations have dealt with the depolymerization and mineralization of natural or synthetic humic acids which did not contain halogen substituents. Thus, depolymerization of humic acids derived from soil or brown coal (lignite) has been described for actinomycetes (Streptomyces viridosporus; Kontchou and Blondeau 1992) and in particular, for various ligninolytic basidiomycetous fungi (Burges and Latter 1960; Blondeau 1989; Dehorter and Blondeau 1992; Ralph and Catcheside 1994; Hofrichter and Fritsche 1996; Willmann and Fakoussa 1997). A partial mineralization of 14C-labeled humic substances was reported for the white-rot fungi Phanerochaete chrysosporium and Trametes versicolor (Dehorter and Blondeau 1993) as well as for isolated Manganese peroxidase from Nematoloma frowardii (Hofrichter et al. 1998). To the best of our knowledge, there is no report about the degradation of halogenated humic acids. Only Lackner et al. (1991)
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have described the degradation of chlorolignins from bleach plant euents by the white-rot fungus Phanerochaete chrysosporium. During our investigation of the metabolism of halophenols in the deuteromycetous soil fungus Penicillium frequentans it became obvious that a spontaneous oligomerization of intermediary halocatechols excreted by the fungus took place (Hofrichter et al. 1994; Wunderwald et al. 1998). These humic acid-like oligomers were not further degraded by P. frequentans. In the present study, we therefore investigated the ability of the humicacid-depolymerizing fungus N. frowardii to convert such substances.
L-4500) ®tted with a HEMA-Bio linear column (Polymer Standard Service, Mainz, Germany) according to a method of Hofrichter and Fritsche (1996, 1997). A UV/VIS spectrometer Shimadzu UV-2102PC was used to determine enzyme activities and the absorbance of FHA-containing solutions. Culture conditions and preparation of manganese peroxidase Cultivation of the agaric white-rot fungus Nematoloma (Hypholoma) frowardii b19 (DSM 11239, ATCC 201144) as well as preparation and characterization of its extracellular manganese peroxidase has been described previously (Schneegass et al. 1997; Hofrichter et al. 1998). Degradation studies
Materials and methods Preparation of synthetic ¯uorinated humic acid-like substance The ¯uorinated humic acid (FHA) was synthesized by spontaneous oxidative polymerization of 3-¯uorocatechol (Aldrich-Chemie, Steinheim, Germany) according to the method of Grieser and Ziechmann (1988) except that 3-¯uorocatechol was used instead of hydroquinone. The aqueous reaction solution contained, in a total of 50 ml, 5 mmol 3-¯uorocatechol and 5 mmol NaOH, and was incubated under aeration at 24 °C on a rotary shaker (140 rpm) for 21 days. The humic acid formed was precipitated with HCl (pH 1.2), centrifuged, washed twice with water, and freeze dried. Physical and chemical analyses 19 F-MAS solid-state NMR spectra were recorded on a 300-MHz NMR spectrometer using a Tecmag console (Tecmag Inc., Houston, Texas, USA). The samples were spun in a 4-mm Bruker MAS (magic angle spinning) probe head at an angle of 54.7° at 10 and 13 kHz; the resonance frequency was 282.41 MHz. The chemical shifts are reported relative to CFCl3. 13 C-CP-TOSS solid-state NMR spectra were recorded on a Bruker ASX 400 spectrometer (Bruker Analytik GmbH, Rheinstetten, Germany). The spinning frequency was 3.5 kHz and the resonance frequency was 100.58 MHz. The chemical shifts are given in relation to adamantane. Infrared measurements were performed with a Paragon 1000 PC Perkin-Elmer FTIR spectrometer. The spectra were recorded from the samples prepared as KBr pellets. Elemental composition (C, H) of the FHA was determined with a LECO CHNS-932 analyzer (LECO Instrumente GmbH, Kirchheim/MuÈnchen, Germany). Carbon-bound ¯uorine was estimated as ¯uoride after UV oxidation (Dubnack 1997). The concentration of ¯uoride was measured with a ¯uoride-sensitive electrode (Orion Model 94-09, Orion Research Incorporated Laboratory Products Group, Boston, USA). The electrode was calibrated with sodium ¯uoride as a standard within a linear concentration range between 10)5 and 10)2 mol/l. The calibration standard was dissolved either in the reaction solution to determine the time course of de¯uorination continuously or was dissolved in deionized water to determine single samples. In the case of single sample measurements, standard solutions and samples were always mixed with TISAB (Total Ion Strength Adjustment Buer) in a ratio of 1:1 to keep ion strength and the pH constant and to complex metal ions with valency above 1. All measurements were carried out in duplicate. Gel permeation chromatography (GPC) was used to detect changes in the molecular mass distribution of the FHA and to follow the formation of lower-molecular-mass fulvic acid-like substances. FHA and fulvic acid-like substances were separated by acid precipitation with HCl prior to GPC analysis. This was carried out using a Merck-Hitachi HPLC apparatus (L-6200/D-2500; Merck, Darmstadt, Germany) with a diode array detector (DAD
In vivo degradation experiments with N. frowardii were carried out in a liquid medium containing 0.25 g l)1 FHA, 5 g l)1 glucose and 0.025 g l)1 MnCl2. Flasks containing 20 ml medium were inoculated with three agar plugs (diameter 9 mm) of active mycelium obtained from 14-day-old malt-extract agar plates. The cultures were incubated at 24 °C on a rotary shaker (140 rpm) in the dark. Samples of 200 ll were centrifuged prior to use and enzyme activities as well as the decrease in absorbance at 450 nm, corresponding to a loss in brown color, were measured every 2±6 days. Fluoride concentration was determined in each ¯ask after ®nishing the experiment on day 24. All values reported are means of triplicate parallel cultivations with standard deviations of less than 10%. Degradation experiments in a cell-free system (in vitro) were carried out in 100-ml ¯asks containing 20 ml of the reaction mixture. The latter consisted of 50 mM sodium malonate buer (pH 4.5), 1 mM MnCl2, 0.6 mM glutathione (GSH), 100 mg l)1 FHA and 500 U l)1 manganese peroxidase (Hofrichter et al. 1998). H2O2 was continuously generated by glucose/glucose oxidase (Aspergillus niger, Sigma-Aldrich GmbH, Deisenhofen, Germany). Reaction mixtures (three ¯asks) were incubated at 37 °C in the dark and stirred continuously. Controls contained boiled manganese peroxidase. Changes of the molecular mass distribution of the acidprecipitable fraction (FHA) and the acid-soluble fraction (fulvic acid-like substances) were controlled by GPC over a period of 75 h. Samples (1 ml) of three ¯asks were combined and prepared for GPC analysis according to Hofrichter and Fritsche (1997). Fluoride concentration was followed continuously over the same period of time. Enzyme assays Manganese peroxidase activity was measured at 270 nm by monitoring the formation of Mn3+-malonate chelates (Wariishi et al. 1992). Activity of laccase was detected with 2,2¢-azinobis(3-ethylbenz-thiazoline-6-sulfonate) (ABTS) according to Eggert et al. (1996).
Results Characterization of the FHA During the preparation of the FHA by oxidative polymerization of 3-¯uorocatechol, a spontaneous de¯uorination of about 20% was observed in relation to the starting content of ¯uorine. The elemental composition of the obtained FHA was 50.4% carbon, 2.6% hydrogen, and 13.6% ¯uorine. From these values an oxygen content of 33.4% was calculated. This value indicates that the polymerization process proceeds via incorpo-
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ration of oxygen, because the oxygen content of the starting material, 3-¯uorocatechol, amounted to only 25%. Analysis of the FHA by solid-state 19F-MAS-NMR gave the following result: the spectrum shows two clear signals, one with an isotropic chemical shift at 29.0 ppm for carbon-bound ¯uorine and one at 13.8 ppm for inorganic ¯uoride (Fig. 1). The latter indicates that in addition to covalent bound ¯uorine, inorganic ¯uoride is somehow attached to the FHA; further quantitative analysis revealed, however, that its concentration was negligible. In comparison, the 19F-MAS-NMR reference spectrum of 3-¯uorocatechol is very similar to that of the FHA showing a signal at 29.3 ppm and an analogous pattern of the spinning side bands (spectrum not given). This gives evidence that the chemical surrounding of the ¯uorine in the FHA formed was not changed much during the polymerization process. The dierence between the two spectra is the missing signal for inorganic ¯uorine and the sharper signal in the spectrum of 3-¯uorocatechol. The slightly broadened spinning side bands of the FHA spectrum are due to its higher amorphism and inhomogeneity in comparison to the starting material. The 13C-CP-TOSS-NMR spectrum shows signals in the region of 112±132 ppm, characteristic of aryl-H and aryl-C carbons, a signal at 146 ppm for aryl-O carbon, and a signal at 169 ppm for carboxyl-carbon (Fig. 2) (Bezile et al. 1997; Knicker 1997; Schmiers et al. 1997). The latter is an indication of the partial splitting of aromatic rings during the polymerization process resulting in the formation of carboxylic groups. This assumption is supported by the infrared spectrum of the FHA showing a band at 1715 cm)1, typical of C O, which was not observed in the infrared spectrum of 3-¯uorocatechol (spectra not shown). The GPC chromatogram of the FHA shows a molecular mass distribution with a maximum of 0.8 kDa (predominant molecular mass) and points to the formation of hexamers. In addition, the shoulder at
Fig. 1 19F-MAS solid-state NMR spectrum of the synthetic ¯uorinated humic acid-like substance (FHA) prepared from 3-¯uorocatechol. The spectrum was recorded at the resonance frequency of 282.41 MHz
Fig. 2 13C-CP-TOSS solid-state NMR spectrum of the synthetic ¯uorinated humic acid-like substance (FHA). The spectrum was recorded at the resonance frequency of 100.58 MHz
0.25 kDa indicates the presence of dimers in the FHA preparation (see Fig. 5A, dashed line). Degradation of FHA by N. frowardii and its manganese peroxidase N. frowardii decolorized the FHA in vivo without adsorption of the material to the fungal hyphae, which were visible due to their light color throughout the experiment. Decolorization due to decreasing pH and subsequently precipitating FHA could be ruled out because neither drastic changes in pH nor the formation of precipitates were observed in the culture liquid. Simultaneously with the decolorization of the medium, increasing manganese peroxidase activity was detectable until the end of incubation on day 24 (Fig. 3), while laccase activity was negligible throughout the experiment and did not exceed 36 U l)1. Manganese peroxidase activity showed a biphasic time course, increasing
Fig. 3 Time courses of manganese peroxidase (.) and laccase activities (r) and decrease in the absorbance at 450 nm (j) during the conversion of FHA (250 mg l)1) by active mycelium of Nematoloma frowardii in liquid culture. The medium contained 0.025 g l)1 MnCl2 (454 lM Mn2+) to stimulate the production of manganese peroxidase. Cultures were incubated over a period of 24 days at 24 °C on a rotary shaker (140 rpm) in the dark
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rapidly between days 6 and 10 and then nearly stagnating at a high level (approximately 400 U l)1) until the 17th day of incubation, after which it increased again reaching a ®nal level of about 750 U l)1. The characteristic biphasic excretion of manganese peroxidase by N. frowardii had already been observed in earlier studies, both in liquid cultures and during solid-state fermentation of wheat straw (Hofrichter and Fritsche 1997; Hofrichter et al. 1999). After the experiment was stopped, a ¯uoride concentration of approximately 1 mM was determined in the culture ¯uid. This corresponds to a release of about 60% of the FHA-bound ¯uorine. Because manganese peroxidase was the predominant ligninolytic enzyme during in vivo degradation of FHA, the potential of the isolated enzyme was tested to convert the FHA in a cell-free system (in vitro). manganese peroxidase was found to depolymerize the FHA with the formation of lower-molecular-mass, fulvic acid-like substances which, unlike the original material, were not precipitable with HCl. The manganese peroxidasecatalyzed depolymerization process was accompanied by the release of about 45% of the bound ¯uorine within 75 h (Fig. 4). During the ®rst 5 h incubation, about 80% of the acid-precipitable FHA was converted; however, the predominant molecular mass of the residual FHA was not changed (Fig. 5A). Simultaneously with the FHA conversion, the acid-soluble fraction (fulvic acidlike substances) increased, accompanied by a slight increase in their predominant molecular mass (Fig. 5B). Control experiments with boiled manganese peroxidase showed only negligible changes in their molecular mass distribution (chromatograms not shown).
Discussion The synthetic ¯uorinated humic acid (FHA) prepared in the present study represents a low-molecular-mass humic acid (predominant molecular mass 0.8 kDa). A similar synthetic humic acid, synthesized from unsubstituted
Fig. 5A, B Gel permeation chromatograms of A the acidprecipitable fractions (humic acids) and B the acid-soluble fractions (fulvic acids) of the FHA during the manganese peroxidase-catalyzed course of depolymerization (starting point: ±±±; after 5 h: -----; after 75 h: áááááá). The reaction conditions are described in Fig. 4
Fig. 4 Time course of de¯uorination during the FHA conversion by the cell-free system of Nematoloma frowardii manganese peroxidase. The reaction mixture contained in a total volume of 20 ml: 50 mM sodium malonate buer (4.5), 1 mM MnCl2, 0.6 mM glutathione, 100 mg l)1 FHA and 500 U l)1 manganese peroxidase. Glucose and glucose oxidase were added to support the action of manganese peroxidase by generating H2O2 at a rate of 20 nmol min)1 ml)1. Reaction mixtures were continuously stirred at 37 °C in the dark
catechol and analyzed by the same GPC method, had a higher predominant molecular mass of 2 kDa (Hofrichter et al. 1998). This dierence in molecular masses is possibly attributable to the addition of heavy metal ions (Cu2+, Fe3+) to the reaction mixture used for the polymerization of catechol. These ions act as catalysts in oxidative polymerization reactions and thus may render the formation of higher-molecular mass humic substances possible (Ziechmann 1994). Liquid cultures of the white-rot fungus N. frowardii degraded the FHA eciently. This degradation became visible by the decolorization of the culture medium, which was originally dark brown. Decolorization (bleaching) of humic acid solutions has been described for several white-rot and litter decaying basidiomycetes.
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Hurst et al. (1960) demonstrated that among a number of basidiomycetous and deuteromycetous fungi tested, only certain white-rot basidiomycetes were capable of bleaching soil humic acids. Hypholoma fasciculare, which is closely related to the N. frowardii used in the present study, was found to be a particularly active humic acid degrader. Similar results were obtained in an extensive screening for coal-humic-acid-depolymerizing fungi comprising nearly 500 fungal strains of dierent taxonomic and ecophysiological groups of fungi. N. frowardii strain was selected as the most active humic acid decomposer (Hofrichter and Fritsche 1996). Furthermore, it was shown that manganese peroxidase of this fungus has the capability to depolymerize natural and synthetic humic acids in a cell-free system (Hofrichter and Fritsche 1997; Hofrichter et al. 1998). The present study demonstrates that a relatively small ¯uorinated humic acid can also be converted in vitro by N. frowardii manganese peroxidase. Some small dierences were observed in the degradation of this FHA compared with higher-molecular mass humic acids. The FHA was degraded without decrease in its molecular mass, which had previously been observed for the residual acid-precipitable fraction of coal humic acids. This is probably attributable to the smallness of the FHA, which enables manganese peroxidase to convert it directly into fulvic acids. The synthetic FHA used probably represents a borderline case between a typical humic acid and a fulvic acid. In this connection, it should be mentioned that the distinction between the two humic substances is arti®cial, so some overlap in their properties is inevitable. 14C-labeled humic acids derived from 14C-catechol, previously used in a comparable study with manganese peroxidase (Hofrichter et al. 1998), were degraded at nearly the same rate as the FHA was, although a four-fold higher enzyme activity was applied. This ®nding shows that even small amounts of manganese peroxidase attack humic substances eectively. The removal of ¯uorine bound in the FHA followed a similar time course as the manganese peroxidase-catalyzed formation of 14CO2 from 14Clabeled humic acids (release of 40% 14CO2 under comparable reaction conditions; Hofrichter et al. 1998). It demonstrates that covalently bound ¯uorine, like aromatic carbon, can be mineralized by manganese peroxidase, i.e., in both cases, inorganic degradation products are formed (F) and CO2, respectively). The present results give indications that halogenated humic substances, for example resulting from the humi®cation of xenobiotic compounds (formation of bound residues), can in principle be eliminated by ligninolytic fungi and their manganese peroxidase system. Moreover, for future investigations the use of ¯uorinated organics as simple model substances to study the decomposition of humic substances appears promising. Acknowledgement We thank the German Ministry for Education and Research (grant 0327051D), which has supported the present work ®nancially.
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