Production of ligninolytic enzymes by Pleurotus sp. and Dichomitus squalens in soil and lignocellulose substrate as influenced by soil microorganisms

Abstract

The activities of the extracellular enzymes laccase and manganese peroxidase (MnP) of the white rot fungi Pleurotus sp. and Dichomitus squalens were measured in the straw substrate and soil layers of solid state cultures. Cultures with sterile soil were compared to cultures with nonsterile soil. For both organisms, enzyme activities in sterile soil were approximately the same as in straw when calculated to the base of the liquid phases (water content) in the straw or soil material. The growth rate and enzyme activities of Pleurotus sp. were not significantly influenced by the presence of soil microorganisms. In contrast, D. squalens did not penetrate the nonsterile soil, and no enzyme activities could be detected in nonsterile soil. Laccase and MnP activities in the straw declined to zero. From this point of view, highly competitive strains like Pleurotus sp. seem to be more suitable for the application in soil remediation than weak competitors (D. squalens) which are not able to maintain appropriate levels of enzyme activities in nonsterile soil.

White rot fungus, Manganese peroxidase, Laccase, Lignocellulose, Soil microorganism, Soil bioremediation

1 Introduction

Lignin degrading or white rot fungi are able to transform and mineralise recalcitrant environmental pollutants such as 4- and 5-ring polycyclic aromatic hydrocarbons [1–5]. Attempts have therefore been made to apply these fungi to the bioremediation of soils contaminated with compounds not sufficiently degradable by other soil microorganisms [6–9]. The initial reactions of degradation by white rot fungi are thought to be catalysed by extracellular ligninolytic enzymes, i.e. oxidases (laccase) and peroxidases [10]. Purified extracellular enzymes are able to transform in vitro compounds such as dibenzodioxine and polycyclic aromatic hydrocarbons [11–15].

If it is accepted that the extracellular ligninolytic enzymes catalyse the critical initial reactions of pollutant transformation, the production and full activity of these enzymes in soil under field conditions are two prerequisites for successful application of white rot fungi in bioremediation. The contaminants are usually poorly water-soluble, but must nevertheless be accessible to the enzymes in the soil matrix. Thus, the fungal hyphae must secrete the enzymes not only into their natural substrate, lignocellulose, but also into the contaminated soil, which is a completely different environment. Also, the enzymes must be produced and remain active in the presence of soil microorganisms. The white rot fungus must grow, or at least survive, and secrete the necessary enzymes under conditions of competition with soil microorganisms.

Most Pleurotus spp. strains tested so far show a highly competitive saprophytic ability against soil microbiota in soil-lignocellulose systems [16–18](see Section 4). In contrast, other white rot basidiomycetes which have been tested were found to be less competitive.

Although the production and activity of ligninolytic enzymes in soil is of great importance for understanding the processes occurring during bioremediation with white rot fungi there are only two mentions of it in the literature [16, 19]. Even data on enzyme production in solid substrates like lignocellulose are scarce [12, 20]. The goal of this study was to determine the activity of extracellular ligninolytic enzymes of white rot fungi in lignocellulose-soil microcosms during different stages of growth, and in the presence and absence of soil microorganisms. We wanted to find out whether the macroscopically visible growth/survival of white rot fungi is an indicator for their extracellular enzyme activity in nonsterile soil, and hence to support criteria for the selection of strains for the application in soil remediation. A competitive Pleurotus sp. strain and a less competitive fungus, Dichomitus squalens, were selected. Both organisms produce laccase and manganese peroxidase (MnP) but no lignin peroxidase.

2 Materials and methods

Pleurotus sp. strain Bhutan (DSM 9618) and D. squalens strain 1b (DSM 9615) were maintained on malt extract agar at 3°C. The soil sample, a luvisol (C_org 0.8%, pH 5.3) from an agricultural site near Braunschweig was sieved to <2 mm, adjusted to 46% of its water holding capacity and left undisturbed at 25°C for 5 days before use. For sterilisation, portions of 11.4 g moist soil were autoclaved twice at 121°C for 20 min with an intervening incubation period of 2 days at 22°C.

Portions of 5 g air-dried, milled (<1 mm) wheat straw were placed in 100 ml conical flasks, moistened with 17 ml deionised water (water content 77.3%) and covered by a nylon mesh. The flasks were closed with cotton plugs and autoclaved at 121°C for 30 min. The straw was inoculated with two discs (diameter 9 mm) cut from 14 days old malt extract agar plate cultures. The cultures were incubated at 22°C until the mycelia had colonised the substrate completely (14 days). Then a layer (4 mm thick) of 11.4 g moist soil (corresponding to 10 g dry matter, water content 12.3%) was distributed on the surface of each straw culture. Half of the flasks were amended with sterile (autoclaved) soil and the other half with native soil. The cotton plugs were replaced by cellophane foil. For each treatment and sampling time, four replicates were run except for the two samplings before addition of soil which were run in triplicate.

For extraction of the enzymes from the culture, the soil layer was collected from the straw surface, which was facilitated by them being separated by a mesh. The complete soil sample was mixed with 8 ml Na-K-phosphate buffer (50 mM, pH 7.0) and extracted on ice for 1 h. The vessels were occasionally shaken by hand during this time. The suspensions were centrifuged at 12000×g for 15 min, the supernatants were collected and centrifuged again. In the supernatant from the second centrifugation step, enzyme activities were measured. The straw cultures colonised by the fungi were cut into small pieces, soaked with 30 ml Na-acetate buffer (160 mM, pH 5.0) and extracted on ice for 3 h with occasional shaking. With an additional 10 ml of buffer, the samples were centrifuged at 8300×g for 15 min. The supernatants were kept on ice until analysed (less than 24 h).

The activities of laccase and manganese peroxidase (MnP) in the straw and soil extracts were determined with 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid, and 3-methyl-2-benzothiazolinone hydrazone and 3-methylaminobenzoic acid, respectively, as substrates with four replicate measurements per sample [16, 21, 22].

Loss of solid organic matter was determined by collecting the solid straw residues after centrifugation, weighing them after drying at 105°C, and calculating the difference against the weight at day 0.

3 Results

The two fungi colonised the straw substrate until week 2 when the soil layers were added. Incubation was continued for another 8 weeks. Pleurotus sp. strain Bhutan grew readily into the soil layers on top of the straw substrate within one week, regardless of whether the soil was sterilised or not. The mycelia appeared more regular in sterile than in nonsterile soil. Fungal hyphae were visible in the sterile and nonsterile soil during the whole incubation period and the straw substrate continuously showed the yellow colour characteristic of white rot.

Dichomitus mycelia was faintly visible in sterile soil 2 weeks after it had been added. Until week 10, the fungal hyphae increasingly linked together straw and soil layer so that it was difficult to remove soil from straw during harvest at weeks 9 and 10. Nonsterile soil was not visibly colonised by Dichomitus mycelia. Instead, the colour of the straw substrate turned from yellow to brown within one week of soil being added. This is the visible consequence of the invasion of the white rot fungus and its substrate by soil microorganisms [18]. The visible change was accompanied by a strong loss of organic matter in the mixed culture as compared to the pure D. squalens culture, during the first two weeks after adding the soil (Fig. 1).

1

Loss of organic matter of the straw compartment during growth of Pleurotus sp. strain Bhutan and Dichomitus squalens in solid state straw-soil cultures in the presence (nonsterile soil, open symbols) and absence (sterile soil, solid symbols) of soil microorganisms. Arrow indicates the time soil was added. Mean values and standard deviations of four replicates are given.

Controls with sterile straw (not colonised by a white rot fungus) plus native soil, that is straw and soil inhabited by soil microorganisms, did not show measurable activities of laccase or MnP at any time. Detection limits were 0.003 U ml⁻¹ for MnP and 0.001 U ml⁻¹ for laccase in straw.

Activities of manganese peroxidase (MnP) were higher than those of laccase for both fungi in both, straw and soil. When comparing the two fungi, Pleurotus sp. produced relatively more laccase and D. squalens more MnP (Fig. 2Fig. 3).

2

Activities of laccase and manganese peroxidase (MnP) in straw during growth of Pleurotus sp. strain Bhutan and Dichomitus squalens in solid state straw-soil cultures in the presence (nonsterile soil, open symbols) and absence (sterile soil, solid symbols) of soil microorganisms. Activities are expressed in units per ml water phase present in the straw or soil compartments, respectively, at the beginning of the experiment. Arrows indicate the time soil was added. Mean values and standard deviations of four replicates are given.

3

Activities of laccase and manganese peroxidase (MnP) in soil. See Fig. 2.

Activities of the two enzymes in straw colonised by Pleurotus steadily increased for 6–7 weeks and then decreased (Fig. 2). In soil, laccase activities reached a distinct maximum (0.6 and 0.8 U ml⁻¹) 2 weeks after addition of the soil (Fig. 3). Neither in straw nor in soil did the presence of soil microorganisms greatly affect enzyme activities. Laccase activities in nonsterile soil tended to be higher than in sterile soil but the differences were not significant for most sampling dates.

In cultures with D. squalens without soil organisms, laccase activities were highest in week 3 in both straw and soil. Activities did not exceed 0.2 U ml⁻¹. In the absence of soil microorganisms, MnP activities in straw and in soil peaked simultaneously in weeks 5 and 8. MnP activities reached 2.9 U ml⁻¹ in straw and 4.0 U ml⁻¹ in soil (Figs. 2 and 3). After the addition of soil with native soil microorganisms, laccase and MnP activities in straw declined to zero within 3 weeks. No enzyme activities were detectable in nonsterile soil at any sampling time (Fig. 3).

4 Discussion

The enzyme activities in the two materials straw and soil depicted in Figs. 2 and 3 are based on the volume of the water phase in the straw or soil, respectively, at the beginning of the experiment, in order to approximate enzyme activities in the biologically relevant watery solution in the undisturbed cultures. A comparison of enzyme activities shows that generally they are not much lower in the soil than in the straw. In fact, sometimes activities were higher in the soil than in the straw, for example, for Pleurotus sp. laccase during weeks 4 and 5, and for D. squalens MnP during weeks 6–8 and for D. squalens laccase during almost all the incubation time. These results suggest that the ligninolytic enzymes are not only secreted in lignin-rich substrates (and transferred to the adjacent soil by diffusion) but are produced by mycelia growing in the soil as well. This means that one important prerequisite for bioremediation by white rot fungi is given: The extracellular ligninolytic enzymes are active in the soil matrix where the pollutants are bound.

Our results show that the extracellular enzymes are only active as long as the white rot fungus is able to compete with the soil microorganisms. The results of this and of former studies [16, 17] reveal that most Pleurotus ssp. are much more competitive against soil microbiota than other white rot fungi, belonging to ten different genera, that have been tested. Pleurotus spp. are able to colonise nonsterile soil [17] and to suppress growth of soil bacteria in their lignocellulose substrate whereas other white rot fungi are not [18]. The determinations of the enzyme activities are in perfect accord with these findings. They suggest that the extracellular enzymes of D. squalens were subject to metabolisation by cohabitating microorganisms as soon as the white rot fungus was unable to compete with soil borne antagonists. In contrast, Pleurotus was able to grow into soil and to maintain the activity of its extracellular enzymes even under the competitive pressure of the soil microorganisms. This comparison of the two fungi shows that the macroscopically visible predominance or dying of the fungus in, or in the presence of, nonsterile soil may be taken as a measure for the activity of their extracellular ligninolytic enzymes in soil. This fact should help in selecting strains of white rot fungi for bioremediation where a long lasting action of the decontaminating catalysts is desired under field, i.e. nonsterile, conditions.

However, our findings do not agree with the mineralisation rate of polycyclic aromatic hydrocarbons (PAHs) by D. squalens and Pleurotus spp. in straw-soil systems [17, 23]– at least not if the hypothesis is accepted that ligninolytic enzymes play a key role during the degradation of organopollutants. Even though D. squalens strain 1b did not secrete laccase or MnP into nonsterile soil, mineralisation of ¹⁴C-PAHs in nonsterile soil added to D. squalens cultures was as high or even higher than in soil added to Pleurotus cultures [17, 23]. The discrepancy between contaminant degradation and enzyme activities found in this study deserves further investigations.

Acknowledgements

We thank A. Gonser for excellent technical assistance and C. in der Wiesche for reading the manuscript. The study was supported by the Deutsche Forschungsgemeinschaft (projects DFG Za 116-2 and 116-3).