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Papers by Jaroslav Šnajdr

The aim of this work was to quantify the effects of vegetation on the activity of extracellular e... more The aim of this work was to quantify the effects of vegetation on the activity of extracellular enzymes in the litter and soil. To achieve this, we investigated a set of post-mining sites in a brown-coal mine deposit area near Sokolov, Czech Republic. The sites were 22e33 years old and had been established on the same initial substrate by planting with six tree genera or leaving for spontaneous revegetation, with four replicate sites per vegetation type. The activity of extracellular hydrolytic and oxidative enzymes and the microbial community composition of the litter and topsoil were compared in the spring, summer and autumn using the dominant tree, pH, soil nutrient content and soil moisture as the explanatory variables. Sites under individual trees exhibited significant differences in the chemical properties of both the litter and soil, and the tree effect was identified as the most important factor affecting the activity of extracellular enzymes either directly or in the interaction with seasonal effects, although not all pairs of tree species were significantly different from each other. Seasonal effects on enzyme activity were only important in the litter. The effects of dominant trees and of seasons contributed equally to the variation in the microbial community composition at individual sites. Only a minor part of the tree effect could be explained by differences in the litter or soil chemistry. Among the chemical variables, the N content most affected the microbial biomass content, increasing fungal (but not bacterial) biomass in the litter and bacterial (but not fungal) biomass in the soil. The results indicate that other factors, such as nutrient quality or the specific association of microorganisms with rhizospheres of different trees or the understory, are likely important mediators of the vegetation effects. When comparing the revegetated sites with sites under spontaneous succession, the enzyme activities and microbial biomass were similar except for the sites revegetated with Alnus which may indicate similar rates of soil development at revegetated and succession sites. Spontaneous succession in temperate Europe may thus be a suitable option for land restoration.

Soil Biology and Biochemistry, 2010

Litter decomposing basidiomycetous fungi produce ligninolytic oxidases and peroxidases which are ... more Litter decomposing basidiomycetous fungi produce ligninolytic oxidases and peroxidases which are involved in the transformation of lignin, as well as humic and fulvic acids. The aim of this work was to evaluate their importance in lignin transformation in forest litter. Two litter decomposing basidiomycete species differing in their abilities to degrade lignin e Hypholoma fasciculare, and Gymnopus erythropus e were cultured on sterile or non-sterile oak litter and their transformation of a 14 C-labelled synthetic lignin (dehydrogenation polymer 14 C-DHP) was compared with that of the indigenous litter microflora. Both in sterile and non-sterile litter, colonisation by basidiomycetes led to higher titres of lignocellulosedegrading enzymes, in particular of laccase and Mn-peroxidase (MnP). The titres of the latter were 6 to 40-fold increased in the presence of basidiomycetes compared to non-sterile litter. During 10 weeks, G. erythropus mineralised over 31% of 14 C-DHP in sterile litter and 23% in non-sterile litter compared to 14% in the non-sterile control. Lignin mineralization by H. fasciculare was comparable to the non-sterile control, 12% in sterile litter and 16% in the non-sterile litter. The largest part of 14 C from 14 C-DHP was transformed into humic compounds during litter treatment with both fungi as well as in the control. In addition to the fast lignin mineralization, microcosms containing G. erythropus also showed a lower final content of unaltered lignin and 23e28% of the lignin was converted into water-soluble compounds with relatively low molecular mass (<5 kDa). Both G. erythropus and H. fasciculare were also able to further mineralise humic compounds. During a 10-week fungal treatment of an artificial 14 C-humic acid ( 14 C-HA) supplemented to the natural humic material of a forest soil, the fungi mineralised 42% and 19% of the labelled material, respectively, under sterile conditions. The 14 C-HA mineralization by introduced basidiomycetes in microcosms containing non-sterile humic material, however, did not significantly differ from that of a non-sterile control and was around 12%. Altogether the results show that saprobic basidiomycetes can considerably differ in their rates of lignin and humic substance conversion. Furthermore, lignin degradation in forest soil can rather slow down by interspecific competition than it is accelerated by cooperation of different microorganisms occupying specific nutritional niches. Therefore, the overall contribution of saprobic basidiomycetes depends on their particular eco-physiological status and the competitive pressure, and may be often lower than initially expected. Significant lignin transformation including partial mineralization is seemingly not exclusively dependent on exceptional high titres of ligninolytic enzymes but also on so far unknown factors. Higher endocellulase production and subsequent weight loss was found in microcosms where saprobic basidiomycetes were combined with indigenous microbes. Potentially, lignin degradation by the basidiomycetes may have increased cellulose availability to the indigenous microbes.

Soil Biology and Biochemistry, 2008

... One unit of enzyme activity was defined as the amount of enzyme releasing 1 ... Phospholipids... more ... One unit of enzyme activity was defined as the amount of enzyme releasing 1 ... Phospholipids were separated using solid-phase extraction cartridges (LiChrolut Si 60, Merck) and the ... Fungal biomass was quantified based on 18:2ω6,9 content (PLFA fung ), bacterial biomass was ...

Soil Biology, 2010

Page 1. Chapter 9 Lignocellulose-Degrading Enzymes in Soils Petr Baldrian and Jaroslav Šnajdr 9.1... more Page 1. Chapter 9 Lignocellulose-Degrading Enzymes in Soils Petr Baldrian and Jaroslav Šnajdr 9.1 Introduction Terrestrial soils contain the largest pool of organic carbon in the biosphere (cca 1,800 Pg). Mineralization of this ...

Soil Biology & Biochemistry, 2008

Changes in the activity of extracellular enzymes (cellobiohydrolase, β-glucosidase, β-xylosidase,... more Changes in the activity of extracellular enzymes (cellobiohydrolase, β-glucosidase, β-xylosidase, chitinase, arylsulfatase and phosphatases) and the changes in microbial community and abiotic properties in the topsoil layer, as well as soil abiotic properties during primary succession were investigated in a brown coal mine deposit area near Sokolov, Czech Republic. The study considered the chronosequence of 4 post-mining plots, 4-, 12-,

The ISME Journal, 2014

Coniferous forests cover extensive areas of the boreal and temperate zones. Owing to their primar... more Coniferous forests cover extensive areas of the boreal and temperate zones. Owing to their primary production and C storage, they have an important role in the global carbon balance. Forest disturbances such as forest fires, windthrows or insect pest outbreaks have a substantial effect on the functioning of these ecosystems. Recent decades have seen an increase in the areas affected by disturbances in both North America and Europe, with indications that this increase is due to both local human activity and global climate change. Here we examine the structural and functional response of the litter and soil microbial community in a Picea abies forest to tree dieback following an invasion of the bark beetle Ips typographus, with a specific focus on the fungal community. The insect-induced disturbance rapidly and profoundly changed vegetation and nutrient availability by killing spruce trees so that the readily available root exudates were replaced by more recalcitrant, polymeric plant biomass components. Owing to the dramatic decrease in photosynthesis, the rate of decomposition processes in the ecosystem decreased as soon as the one-time litter input had been processed. The fungal community showed profound changes, including a decrease in biomass (2.5-fold in the litter and 12-fold in the soil) together with the disappearance of fungi symbiotic with tree roots and a relative increase in saprotrophic taxa. Within the latter group, successive changes reflected the changing availability of needle litter and woody debris. Bacterial biomass appeared to be either unaffected or increased after the disturbance, resulting in a substantial increase in the bacterial/fungal biomass ratio.

Research in Microbiology, 2007

Due to production of lignocellulose-degrading enzymes, saprotrophic litter-decomposing basidiomyc... more Due to production of lignocellulose-degrading enzymes, saprotrophic litter-decomposing basidiomycetes can significantly contribute to the turnover of soil organic matter. The production of lignin and polysaccharide-degrading enzymes and changes in the chemical composition of litter was studied with Marasmius quercophilus, Mycena inclinata and Pholiota lenta, three basidiomycete species typical of oak (Quercus petraea) forests. Within 12 weeks of incubation, M. inclinata decomposed 33%, M. quercophilus 36% and P. lenta 48% of the substrate dry mass. All fungi produced laccase and Mn-peroxidase and none of them produced lignin peroxidase or Mn-independent peroxidases. M. inclinata and M. quercophilus produced considerable laccase activity, while production by P. lenta was low. M. quercophilus and P. lenta produced most Mn-peroxidase at the beginning of the experiment, while the production by M. inclinata was more stable in time. Endo-1,4-b-xylanase exhibited the highest activity among endocleaving glycosyl hydrolases while 1,4-b-glucosidase was the main exocleaving enzyme. All fungi decreased the C:N ratio of the litter from 27 to 13e17 and M. inclinata and M. quercophilus also decreased the lignin content. Analytical pyrolysis of decayed litter showed changes in litter composition similar to those caused by white-rot fungi during wood decay, e.g. a decrease in the syringyl/guaiacyl lignin ratio. These changes were more pronounced in M. inclinata and M. quercophilus. The results indicate that different litter-decomposing fungi can cause substantial litter transformation despite considerable differences in the production of lignocellulose-degrading enzymes.

Fungal Ecology, 2011

Chemical composition of litter has previously been reported to affect in situ decomposition. To i... more Chemical composition of litter has previously been reported to affect in situ decomposition. To identify its effects on a single species level, the saprotrophic basidiomycete Hypholoma fasciculare was grown on 11 types of litter with variable chemical composition (N content of 3.4–28.9mgg−1), and the mass loss of litter and lignin, production of extracellular enzymes and fungal biomass were followed. After 12 weeks, mass loss ranged from 16% to 34%. During early decomposition stages, litter mass loss, fungal biomass production (estimated by ergosterol content) as well as fungal substrate use efficiency all increased with increasing initial N content of the litter. The initial litter decomposition rate was significantly positively correlated with the activities of arylsulfatase, cellobiohydrolase, endoxylanase and phosphatase. Contrary to expectations, the lignin content did not affect litter mass loss, when covariation with N content was accounted for. The ratio of lignin loss to total mass loss depended on the litter type and did not reflect the activities of ligninolytic enzymes.

Folia Microbiologica, 2006

The extracellular enzyme activity and changes in soil bacterial community during the growth of th... more The extracellular enzyme activity and changes in soil bacterial community during the growth of the ligninolytic fungus Pleurotus ostreatus were determined in nonsterile soil with low and high available carbon content. In soil with P. ostreatus, the activity of ligninolytic enzymes laccase and Mn-peroxidase was several orders of magnitude higher than in soil without the fungus. Addition of lignocellulose to soil increased the activity of cellulolytic fungi and the production of Mn-peroxidase by P. ostreatus. The counts of heterotrophic bacteria were more significantly affected by the presence of lignocellulose than by P. ostreatus. The effects of both substrate addition and time (succession) were more significant factors affecting the soil bacterial community than the presence of P. ostreatus. Bacterial community structure was affected by fungal colonization in low carbon soil, where a decrease of diversity and changes in substrate utilization profiles were detected.

Folia Microbiologica, 2007

Enzyme activity was determined in cultures of Pleurotus ostreatus and Trametes versicolor with ce... more Enzyme activity was determined in cultures of Pleurotus ostreatus and Trametes versicolor with cellulose as a sole C source and high C/N ratio. The fungi were able to grow and produce laccase and Mn-peroxidase (MnP) at 5-35 °C, the highest production being recorded at 25-30 °C in P. ostreatus and at 35 °C in T. versicolor. Production of both enzymes at 10 °C accounted only for 4-20 % of the maximum value. Temperature optima for enzyme activity were 50 and 55 °C for P. ostreatus and T. versicolor laccases, respectively, and 60 °C for MnP. Temperatures causing 50 % loss of activity after 24 h were 32 and 47 °C for laccases and 36 and 30 °C for MnP from P. ostreatus and T. versicolor, respectively.

FEMS Microbiology Ecology, 2011

Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates ... more Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates in soil. The production of extracellular hydrolytic enzymes was studied during the growth of two saprotrophic basidiomycetes, Hypholoma fasciculare and Phanerochaete velutina, across the surface of nonsterile soil microcosms, along with the effects of these basidiomycetes on fungi and bacteria within the soil. Higher activities of a-glucosidase, b-glucosidase, cellobiohydrolase, bxylosidase, phosphomonoesterase and phosphodiesterase, but not of arylsulphatase, were recorded beneath the mycelia. Despite the fact that H. fasciculare, with exploitative hyphal growth, produced much denser hyphal cover on the soil surface than P. velutina, with explorative growth, both fungi produced similar amounts of extracellular enzymes. In the areas where the mycelia of H. fasciculare and P. velutina interacted, the activities of N-acetylglucosaminidase, a-glucosidase and phosphomonoesterase, the enzymes potentially involved in hyphal cell wall damage, and the utilization of compounds released from damaged hyphae of interacting fungi, were particularly increased. No significant differences in fungal biomass were observed between basidiomycete-colonized and noncolonized soil, but bacterial biomass was reduced in soil with H. fasciculare. The increases in the activities of b-xylosidase, b-glucosidase, phosphomonoesterase and cellobiohydrolase with increasing fungal : bacterial biomass ratio indicate the positive effects of fungal enzymes on nutrient release and bacterial abundance, which is reflected in the positive correlation of bacterial and fungal biomass content.

FEMS Microbiology Ecology, 2011

The links among the changes in litter chemistry, the activity of extracellular enzymes and the mi... more The links among the changes in litter chemistry, the activity of extracellular enzymes and the microbial community composition were observed in Quercus petraea litter. Three phases of decomposition could be distinguished. In the early 4month stage, with high activities of b-glucosidase, b-xylosidase and cellobiohydrolase, 16.4% of litter was decomposed. Hemicelluloses were rapidly removed while cellulose and lignin degradation was slow. In months 4-12, with high endocellulase and endoxylanase activities, decomposition of cellulose prevailed and 31.8% of litter mass was lost. After the third phase of decomposition until month 24 with high activity of ligninolytic enzymes, the litter mass loss reached 67.9%. After 2 years of decay, cellulose decomposition was almost complete and most of the remaining polysaccharides were in the form of hemicelluloses. Fungi largely dominated over bacteria as leaf endophytes and also in the litter immediately before contact with soil, and this fungal dominance lasted until month 4. Bacterial biomass (measured as phospholipid fatty acid content) in litter increased with time but also changed qualitatively, showing an increasing number of Actinobacteria. This paper shows that the dynamics of decomposition of individual litter components changes with time in accordance with the changes in the microbial community composition and its production of extracellular enzymes.

Enzyme and Microbial Technology, 2006

Litter-decomposing basidiomycete fungi (LDF) including environmental isolates from oak forest soi... more Litter-decomposing basidiomycete fungi (LDF) including environmental isolates from oak forest soil were compared with white-rot fungi for ligninolytic enzymes production and decolorization of synthetic dyes Poly B-411, Reactive Black 5, Reactive Orange 16 and Remazol Brilliant Blue R (RBBR). LDF differed significantly in laccase production. Mycena inclinata and Collybia dryophila produced significant amounts of the enzyme during the whole experiment, while the production in Stropharia rugosoannulata started after 3 weeks of cultivation. Soil isolates exhibited detectable though very low laccase activity. The highest activity of Mn-peroxidase was detected in the cultures of C. dryophila with the peak activities over 30 U l −1 . In all other strains, Mn-peroxidase activity did not exceed 3 U l −1 . The decolorization of 100 mg l −1 dyes after 28 days ranged 80-95% for RBBR, 60-95% for Poly B-411, 58-85% for Reactive Black 5 and 45-82% for Reactive Orange 16. The fastest degradation of Poly B-411 was performed by the strains with high levels of laccase and MnP while the decolorization of other dyes did not depend so strictly on enzyme activities. The highest decolorization of azo dyes was achieved with the LDF C. dryophila, S. rugosoannulata and the soil isolates. The presence of dyes significantly affected enzyme activities in fungal cultures.

Enzyme and Microbial Technology, 2008

Activity and production of extracellular enzymes by saprotrophic litter-decomposing basidiomycete... more Activity and production of extracellular enzymes by saprotrophic litter-decomposing basidiomycetes Hypholoma fasciculare and Rhodocollybia butyracea was studied in microcosms with reconstructed L, O and Ah horizons of a soil profile of Quercus petraea forest soil. Both H. fasciculare and R. butyracea colonized the L layer of microcosms rapidly, while the colonization of O layer was slower. The Ah layer was substantially colonized only by R. butyracea. Enzyme activities in the soil microcosms decreased from the L layer > O layer > Ah layer and activities in microcosms inoculated with the fungi were quite similar to each other. Compared to control, the most apparent was the increase of ligninolytic enzyme activities. Laccase activities in H. fasciculare and R. butyracea-colonized L layers were 3-fold compared to control and the activity maxima of Mnperoxidase in fungus-colonized O layers were 2-3-fold and in the L layers up to 40-fold compared to controls. Activities of cellulolytic enzymes, chitinase and acidic phosphatase in both fungal treatments were higher in the L layer on weeks 2-6 while the activity of alkaline phosphatase did not show differences between fungus-colonized and control treatments. Both fungi decreased fungal CFU in the L layer but significantly increased the counts in the O layer. Both fungi also increased bacterial CFU in the O layer, R. butyracea more than H. fasciculare. The analysis of fungal and bacterial biomass based on ergosterol content and PLFA analysis showed a sharp decrease from L to Ah layer, but did not show significant differences among treatments.

Chemosphere, 2012

a b s t r a c t Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are copper-containing... more a b s t r a c t Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are copper-containing enzymes that catalyze the oxidative conversion of a variety of chemicals, such as mono-, oligo-, and polyphenols and aromatic amines. Laccases have been proposed to participate in the transformation of organic matter and xenobiotics as well as microbial interactions. Several laccase assays have been proposed and used in soils. Here, we show that the optimal pH conditions for the laccase substrates 2,2 0 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, pH 3-5), 2,6-dimethoxyphenol (4-5.5), L-3,4-dihydroxyphenylalanine (DOPA; 4-6), guaiacol (3.5-5), 4-methylcatechol (3.5-5), and syringaldazine (5.5-7.0) are similar between purified laccases from Trametes versicolor and Pyricularia sp. and soil extracts; the substrate affinities of purified enzymes (K M ) and soil extracts were also similar. The laccase assays showed specificity overlap with tyrosinase and ligninolytic peroxidases when hydrogen peroxide is present. The ABTS oxidation assay is able to reliably detect the presence of 13.5 pg mL À1 or 0.199 Â 10 À12 mol mL À1 of T. versicolor laccase, which is three times more sensitive than the 2,6-dimethoxyphenol-based assay and more than 40 times more sensitive than any of the other assays. The low molecular mass soil-derived compounds and the isolated fulvic and humic acids influence the laccase assays and should be removed from the soil extracts before measurements of the enzyme activity are performed.

Biology and Fertility of Soils, 2010

The small-scale distribution of activities of extracellular laccase, Mn-peroxidase, endoglucanase... more The small-scale distribution of activities of extracellular laccase, Mn-peroxidase, endoglucanase, cellobiohydrolase, β-glucosidase, endoxylanase, βxylosidase, chitinase, and acid phosphatase were studied in the litter (L) and organic (H) horizons of Quercus petraea forest soil and related to the distribution of microbial biomass. Geostatistical analysis showed that the spatial autocorrelation of the enzyme activities and soil microbial biomass measured as phospholipid fatty acids (PLFA) and ergosterol content occurred at similar scales, typically in the range of tens of centimeters. The size of the spatial structures differed between the L and H horizons; for most of the studied enzymatic processes, litter exhibited a higher spatial variability (smaller autocorrelation distances). The distribution of several enzymes, including laccase, Mn-peroxidase, and some hydrolases, reflected the distribution of fungal biomass. Polysaccharide hydrolases exhibited similar spatial distribution patterns in the L horizon, and their activity coincided with a high fungal/ bacterial biomass ratio.

Biodegradation, 2011

Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cell... more Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cellulose in dead wood and as such they attracted considerable attention. The aims of this work were to quantify the activity and spatial distribution of extracellular enzymes in coarse wood colonised by the white-rot basidiomycete Fomes fomentarius and in adjacent fruitbodies of the fungus and to analyse the diversity of the fungal and bacterial community in a fungus-colonised wood and its potential effect on enzyme production by F. fomentarius. Fungus-colonised wood and fruitbodies were collected in low management intensity forests in the Czech Republic. There were significant differences in enzyme production by F. fomentarius between Betula pendula and Fagus sylvatica wood, the activity of cellulose and xylan-degrading enzymes was significantly higher in beech wood than in birch wood. Spatial analysis of a sample B. pendula log segment proved that F. fomentarius was the single fungal representative found in the log. There was a high level of spatial variability in the amount of fungal biomass detected, but no effects on enzyme activities were observed. Samples from the fruiting body showed high b-glucosidase and chitinase activities compared to wood samples. Significantly higher levels of xylanase and cellobiohydrolase were found in samples located near the fruitbody (proximal), and higher laccase and Mn-peroxidase activities were found in the distal ones. The microbial community in wood was dominated by the fungus (fungal to bacterial DNA ratio of 62-111). Bacterial abundance composition was lower in proximal than distal parts of wood by a factor of 24. These results show a significant level of spatial heterogeneity in coarse wood. One of the explanations may be the successive colonization of wood by the fungus: due to differential enzyme production, the rates of biodegradation of coarse wood are also spatially inhomogeneous.

Applied Soil Ecology, 2010

The ISME Journal, 2012

Soils of coniferous forest ecosystems are important for the global carbon cycle, and the identifi... more Soils of coniferous forest ecosystems are important for the global carbon cycle, and the identification of active microbial decomposers is essential for understanding organic matter transformation in these ecosystems. By the independent analysis of DNA and RNA, whole communities of bacteria and fungi and its active members were compared in topsoil of a Picea abies forest during a period of organic matter decomposition. Fungi quantitatively dominate the microbial community in the litter horizon, while the organic horizon shows comparable amount of fungal and bacterial biomasses. Active microbial populations obtained by RNA analysis exhibit similar diversity as DNA-derived populations, but significantly differ in the composition of microbial taxa. Several highly active taxa, especially fungal ones, show low abundance or even absence in the DNA pool. Bacteria and especially fungi are often distinctly associated with a particular soil horizon. Fungal communities are less even than bacterial ones and show higher relative abundances of dominant species. While dominant bacterial species are distributed across the studied ecosystem, distribution of dominant fungi is often spatially restricted as they are only recovered at some locations. The sequences of cbhI gene encoding for cellobiohydrolase (exocellulase), an essential enzyme for cellulose decomposition, were compared in soil metagenome and metatranscriptome and assigned to their producers. Litter horizon exhibits higher diversity and higher proportion of expressed sequences than organic horizon. Cellulose decomposition is mediated by highly diverse fungal populations largely distinct between soil horizons. The results indicate that low-abundance species make an important contribution to decomposition processes in soils.

The aim of this work was to quantify the effects of vegetation on the activity of extracellular e... more The aim of this work was to quantify the effects of vegetation on the activity of extracellular enzymes in the litter and soil. To achieve this, we investigated a set of post-mining sites in a brown-coal mine deposit area near Sokolov, Czech Republic. The sites were 22e33 years old and had been established on the same initial substrate by planting with six tree genera or leaving for spontaneous revegetation, with four replicate sites per vegetation type. The activity of extracellular hydrolytic and oxidative enzymes and the microbial community composition of the litter and topsoil were compared in the spring, summer and autumn using the dominant tree, pH, soil nutrient content and soil moisture as the explanatory variables. Sites under individual trees exhibited significant differences in the chemical properties of both the litter and soil, and the tree effect was identified as the most important factor affecting the activity of extracellular enzymes either directly or in the interaction with seasonal effects, although not all pairs of tree species were significantly different from each other. Seasonal effects on enzyme activity were only important in the litter. The effects of dominant trees and of seasons contributed equally to the variation in the microbial community composition at individual sites. Only a minor part of the tree effect could be explained by differences in the litter or soil chemistry. Among the chemical variables, the N content most affected the microbial biomass content, increasing fungal (but not bacterial) biomass in the litter and bacterial (but not fungal) biomass in the soil. The results indicate that other factors, such as nutrient quality or the specific association of microorganisms with rhizospheres of different trees or the understory, are likely important mediators of the vegetation effects. When comparing the revegetated sites with sites under spontaneous succession, the enzyme activities and microbial biomass were similar except for the sites revegetated with Alnus which may indicate similar rates of soil development at revegetated and succession sites. Spontaneous succession in temperate Europe may thus be a suitable option for land restoration.

Soil Biology and Biochemistry, 2010

Litter decomposing basidiomycetous fungi produce ligninolytic oxidases and peroxidases which are ... more Litter decomposing basidiomycetous fungi produce ligninolytic oxidases and peroxidases which are involved in the transformation of lignin, as well as humic and fulvic acids. The aim of this work was to evaluate their importance in lignin transformation in forest litter. Two litter decomposing basidiomycete species differing in their abilities to degrade lignin e Hypholoma fasciculare, and Gymnopus erythropus e were cultured on sterile or non-sterile oak litter and their transformation of a 14 C-labelled synthetic lignin (dehydrogenation polymer 14 C-DHP) was compared with that of the indigenous litter microflora. Both in sterile and non-sterile litter, colonisation by basidiomycetes led to higher titres of lignocellulosedegrading enzymes, in particular of laccase and Mn-peroxidase (MnP). The titres of the latter were 6 to 40-fold increased in the presence of basidiomycetes compared to non-sterile litter. During 10 weeks, G. erythropus mineralised over 31% of 14 C-DHP in sterile litter and 23% in non-sterile litter compared to 14% in the non-sterile control. Lignin mineralization by H. fasciculare was comparable to the non-sterile control, 12% in sterile litter and 16% in the non-sterile litter. The largest part of 14 C from 14 C-DHP was transformed into humic compounds during litter treatment with both fungi as well as in the control. In addition to the fast lignin mineralization, microcosms containing G. erythropus also showed a lower final content of unaltered lignin and 23e28% of the lignin was converted into water-soluble compounds with relatively low molecular mass (<5 kDa). Both G. erythropus and H. fasciculare were also able to further mineralise humic compounds. During a 10-week fungal treatment of an artificial 14 C-humic acid ( 14 C-HA) supplemented to the natural humic material of a forest soil, the fungi mineralised 42% and 19% of the labelled material, respectively, under sterile conditions. The 14 C-HA mineralization by introduced basidiomycetes in microcosms containing non-sterile humic material, however, did not significantly differ from that of a non-sterile control and was around 12%. Altogether the results show that saprobic basidiomycetes can considerably differ in their rates of lignin and humic substance conversion. Furthermore, lignin degradation in forest soil can rather slow down by interspecific competition than it is accelerated by cooperation of different microorganisms occupying specific nutritional niches. Therefore, the overall contribution of saprobic basidiomycetes depends on their particular eco-physiological status and the competitive pressure, and may be often lower than initially expected. Significant lignin transformation including partial mineralization is seemingly not exclusively dependent on exceptional high titres of ligninolytic enzymes but also on so far unknown factors. Higher endocellulase production and subsequent weight loss was found in microcosms where saprobic basidiomycetes were combined with indigenous microbes. Potentially, lignin degradation by the basidiomycetes may have increased cellulose availability to the indigenous microbes.

Soil Biology and Biochemistry, 2008

... One unit of enzyme activity was defined as the amount of enzyme releasing 1 ... Phospholipids... more ... One unit of enzyme activity was defined as the amount of enzyme releasing 1 ... Phospholipids were separated using solid-phase extraction cartridges (LiChrolut Si 60, Merck) and the ... Fungal biomass was quantified based on 18:2ω6,9 content (PLFA fung ), bacterial biomass was ...

Soil Biology, 2010

Page 1. Chapter 9 Lignocellulose-Degrading Enzymes in Soils Petr Baldrian and Jaroslav Šnajdr 9.1... more Page 1. Chapter 9 Lignocellulose-Degrading Enzymes in Soils Petr Baldrian and Jaroslav Šnajdr 9.1 Introduction Terrestrial soils contain the largest pool of organic carbon in the biosphere (cca 1,800 Pg). Mineralization of this ...

Soil Biology & Biochemistry, 2008

Changes in the activity of extracellular enzymes (cellobiohydrolase, β-glucosidase, β-xylosidase,... more Changes in the activity of extracellular enzymes (cellobiohydrolase, β-glucosidase, β-xylosidase, chitinase, arylsulfatase and phosphatases) and the changes in microbial community and abiotic properties in the topsoil layer, as well as soil abiotic properties during primary succession were investigated in a brown coal mine deposit area near Sokolov, Czech Republic. The study considered the chronosequence of 4 post-mining plots, 4-, 12-,

The ISME Journal, 2014

Coniferous forests cover extensive areas of the boreal and temperate zones. Owing to their primar... more Coniferous forests cover extensive areas of the boreal and temperate zones. Owing to their primary production and C storage, they have an important role in the global carbon balance. Forest disturbances such as forest fires, windthrows or insect pest outbreaks have a substantial effect on the functioning of these ecosystems. Recent decades have seen an increase in the areas affected by disturbances in both North America and Europe, with indications that this increase is due to both local human activity and global climate change. Here we examine the structural and functional response of the litter and soil microbial community in a Picea abies forest to tree dieback following an invasion of the bark beetle Ips typographus, with a specific focus on the fungal community. The insect-induced disturbance rapidly and profoundly changed vegetation and nutrient availability by killing spruce trees so that the readily available root exudates were replaced by more recalcitrant, polymeric plant biomass components. Owing to the dramatic decrease in photosynthesis, the rate of decomposition processes in the ecosystem decreased as soon as the one-time litter input had been processed. The fungal community showed profound changes, including a decrease in biomass (2.5-fold in the litter and 12-fold in the soil) together with the disappearance of fungi symbiotic with tree roots and a relative increase in saprotrophic taxa. Within the latter group, successive changes reflected the changing availability of needle litter and woody debris. Bacterial biomass appeared to be either unaffected or increased after the disturbance, resulting in a substantial increase in the bacterial/fungal biomass ratio.

Research in Microbiology, 2007

Due to production of lignocellulose-degrading enzymes, saprotrophic litter-decomposing basidiomyc... more Due to production of lignocellulose-degrading enzymes, saprotrophic litter-decomposing basidiomycetes can significantly contribute to the turnover of soil organic matter. The production of lignin and polysaccharide-degrading enzymes and changes in the chemical composition of litter was studied with Marasmius quercophilus, Mycena inclinata and Pholiota lenta, three basidiomycete species typical of oak (Quercus petraea) forests. Within 12 weeks of incubation, M. inclinata decomposed 33%, M. quercophilus 36% and P. lenta 48% of the substrate dry mass. All fungi produced laccase and Mn-peroxidase and none of them produced lignin peroxidase or Mn-independent peroxidases. M. inclinata and M. quercophilus produced considerable laccase activity, while production by P. lenta was low. M. quercophilus and P. lenta produced most Mn-peroxidase at the beginning of the experiment, while the production by M. inclinata was more stable in time. Endo-1,4-b-xylanase exhibited the highest activity among endocleaving glycosyl hydrolases while 1,4-b-glucosidase was the main exocleaving enzyme. All fungi decreased the C:N ratio of the litter from 27 to 13e17 and M. inclinata and M. quercophilus also decreased the lignin content. Analytical pyrolysis of decayed litter showed changes in litter composition similar to those caused by white-rot fungi during wood decay, e.g. a decrease in the syringyl/guaiacyl lignin ratio. These changes were more pronounced in M. inclinata and M. quercophilus. The results indicate that different litter-decomposing fungi can cause substantial litter transformation despite considerable differences in the production of lignocellulose-degrading enzymes.

Fungal Ecology, 2011

Chemical composition of litter has previously been reported to affect in situ decomposition. To i... more Chemical composition of litter has previously been reported to affect in situ decomposition. To identify its effects on a single species level, the saprotrophic basidiomycete Hypholoma fasciculare was grown on 11 types of litter with variable chemical composition (N content of 3.4–28.9mgg−1), and the mass loss of litter and lignin, production of extracellular enzymes and fungal biomass were followed. After 12 weeks, mass loss ranged from 16% to 34%. During early decomposition stages, litter mass loss, fungal biomass production (estimated by ergosterol content) as well as fungal substrate use efficiency all increased with increasing initial N content of the litter. The initial litter decomposition rate was significantly positively correlated with the activities of arylsulfatase, cellobiohydrolase, endoxylanase and phosphatase. Contrary to expectations, the lignin content did not affect litter mass loss, when covariation with N content was accounted for. The ratio of lignin loss to total mass loss depended on the litter type and did not reflect the activities of ligninolytic enzymes.

Folia Microbiologica, 2006

The extracellular enzyme activity and changes in soil bacterial community during the growth of th... more The extracellular enzyme activity and changes in soil bacterial community during the growth of the ligninolytic fungus Pleurotus ostreatus were determined in nonsterile soil with low and high available carbon content. In soil with P. ostreatus, the activity of ligninolytic enzymes laccase and Mn-peroxidase was several orders of magnitude higher than in soil without the fungus. Addition of lignocellulose to soil increased the activity of cellulolytic fungi and the production of Mn-peroxidase by P. ostreatus. The counts of heterotrophic bacteria were more significantly affected by the presence of lignocellulose than by P. ostreatus. The effects of both substrate addition and time (succession) were more significant factors affecting the soil bacterial community than the presence of P. ostreatus. Bacterial community structure was affected by fungal colonization in low carbon soil, where a decrease of diversity and changes in substrate utilization profiles were detected.

Folia Microbiologica, 2007

Enzyme activity was determined in cultures of Pleurotus ostreatus and Trametes versicolor with ce... more Enzyme activity was determined in cultures of Pleurotus ostreatus and Trametes versicolor with cellulose as a sole C source and high C/N ratio. The fungi were able to grow and produce laccase and Mn-peroxidase (MnP) at 5-35 °C, the highest production being recorded at 25-30 °C in P. ostreatus and at 35 °C in T. versicolor. Production of both enzymes at 10 °C accounted only for 4-20 % of the maximum value. Temperature optima for enzyme activity were 50 and 55 °C for P. ostreatus and T. versicolor laccases, respectively, and 60 °C for MnP. Temperatures causing 50 % loss of activity after 24 h were 32 and 47 °C for laccases and 36 and 30 °C for MnP from P. ostreatus and T. versicolor, respectively.

FEMS Microbiology Ecology, 2011

Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates ... more Saprotrophic cord-forming basidiomycetes are important decomposers of lignocellulosic substrates in soil. The production of extracellular hydrolytic enzymes was studied during the growth of two saprotrophic basidiomycetes, Hypholoma fasciculare and Phanerochaete velutina, across the surface of nonsterile soil microcosms, along with the effects of these basidiomycetes on fungi and bacteria within the soil. Higher activities of a-glucosidase, b-glucosidase, cellobiohydrolase, bxylosidase, phosphomonoesterase and phosphodiesterase, but not of arylsulphatase, were recorded beneath the mycelia. Despite the fact that H. fasciculare, with exploitative hyphal growth, produced much denser hyphal cover on the soil surface than P. velutina, with explorative growth, both fungi produced similar amounts of extracellular enzymes. In the areas where the mycelia of H. fasciculare and P. velutina interacted, the activities of N-acetylglucosaminidase, a-glucosidase and phosphomonoesterase, the enzymes potentially involved in hyphal cell wall damage, and the utilization of compounds released from damaged hyphae of interacting fungi, were particularly increased. No significant differences in fungal biomass were observed between basidiomycete-colonized and noncolonized soil, but bacterial biomass was reduced in soil with H. fasciculare. The increases in the activities of b-xylosidase, b-glucosidase, phosphomonoesterase and cellobiohydrolase with increasing fungal : bacterial biomass ratio indicate the positive effects of fungal enzymes on nutrient release and bacterial abundance, which is reflected in the positive correlation of bacterial and fungal biomass content.

FEMS Microbiology Ecology, 2011

The links among the changes in litter chemistry, the activity of extracellular enzymes and the mi... more The links among the changes in litter chemistry, the activity of extracellular enzymes and the microbial community composition were observed in Quercus petraea litter. Three phases of decomposition could be distinguished. In the early 4month stage, with high activities of b-glucosidase, b-xylosidase and cellobiohydrolase, 16.4% of litter was decomposed. Hemicelluloses were rapidly removed while cellulose and lignin degradation was slow. In months 4-12, with high endocellulase and endoxylanase activities, decomposition of cellulose prevailed and 31.8% of litter mass was lost. After the third phase of decomposition until month 24 with high activity of ligninolytic enzymes, the litter mass loss reached 67.9%. After 2 years of decay, cellulose decomposition was almost complete and most of the remaining polysaccharides were in the form of hemicelluloses. Fungi largely dominated over bacteria as leaf endophytes and also in the litter immediately before contact with soil, and this fungal dominance lasted until month 4. Bacterial biomass (measured as phospholipid fatty acid content) in litter increased with time but also changed qualitatively, showing an increasing number of Actinobacteria. This paper shows that the dynamics of decomposition of individual litter components changes with time in accordance with the changes in the microbial community composition and its production of extracellular enzymes.

Enzyme and Microbial Technology, 2006

Litter-decomposing basidiomycete fungi (LDF) including environmental isolates from oak forest soi... more Litter-decomposing basidiomycete fungi (LDF) including environmental isolates from oak forest soil were compared with white-rot fungi for ligninolytic enzymes production and decolorization of synthetic dyes Poly B-411, Reactive Black 5, Reactive Orange 16 and Remazol Brilliant Blue R (RBBR). LDF differed significantly in laccase production. Mycena inclinata and Collybia dryophila produced significant amounts of the enzyme during the whole experiment, while the production in Stropharia rugosoannulata started after 3 weeks of cultivation. Soil isolates exhibited detectable though very low laccase activity. The highest activity of Mn-peroxidase was detected in the cultures of C. dryophila with the peak activities over 30 U l −1 . In all other strains, Mn-peroxidase activity did not exceed 3 U l −1 . The decolorization of 100 mg l −1 dyes after 28 days ranged 80-95% for RBBR, 60-95% for Poly B-411, 58-85% for Reactive Black 5 and 45-82% for Reactive Orange 16. The fastest degradation of Poly B-411 was performed by the strains with high levels of laccase and MnP while the decolorization of other dyes did not depend so strictly on enzyme activities. The highest decolorization of azo dyes was achieved with the LDF C. dryophila, S. rugosoannulata and the soil isolates. The presence of dyes significantly affected enzyme activities in fungal cultures.

Enzyme and Microbial Technology, 2008

Activity and production of extracellular enzymes by saprotrophic litter-decomposing basidiomycete... more Activity and production of extracellular enzymes by saprotrophic litter-decomposing basidiomycetes Hypholoma fasciculare and Rhodocollybia butyracea was studied in microcosms with reconstructed L, O and Ah horizons of a soil profile of Quercus petraea forest soil. Both H. fasciculare and R. butyracea colonized the L layer of microcosms rapidly, while the colonization of O layer was slower. The Ah layer was substantially colonized only by R. butyracea. Enzyme activities in the soil microcosms decreased from the L layer > O layer > Ah layer and activities in microcosms inoculated with the fungi were quite similar to each other. Compared to control, the most apparent was the increase of ligninolytic enzyme activities. Laccase activities in H. fasciculare and R. butyracea-colonized L layers were 3-fold compared to control and the activity maxima of Mnperoxidase in fungus-colonized O layers were 2-3-fold and in the L layers up to 40-fold compared to controls. Activities of cellulolytic enzymes, chitinase and acidic phosphatase in both fungal treatments were higher in the L layer on weeks 2-6 while the activity of alkaline phosphatase did not show differences between fungus-colonized and control treatments. Both fungi decreased fungal CFU in the L layer but significantly increased the counts in the O layer. Both fungi also increased bacterial CFU in the O layer, R. butyracea more than H. fasciculare. The analysis of fungal and bacterial biomass based on ergosterol content and PLFA analysis showed a sharp decrease from L to Ah layer, but did not show significant differences among treatments.

Chemosphere, 2012

a b s t r a c t Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are copper-containing... more a b s t r a c t Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are copper-containing enzymes that catalyze the oxidative conversion of a variety of chemicals, such as mono-, oligo-, and polyphenols and aromatic amines. Laccases have been proposed to participate in the transformation of organic matter and xenobiotics as well as microbial interactions. Several laccase assays have been proposed and used in soils. Here, we show that the optimal pH conditions for the laccase substrates 2,2 0 -azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, pH 3-5), 2,6-dimethoxyphenol (4-5.5), L-3,4-dihydroxyphenylalanine (DOPA; 4-6), guaiacol (3.5-5), 4-methylcatechol (3.5-5), and syringaldazine (5.5-7.0) are similar between purified laccases from Trametes versicolor and Pyricularia sp. and soil extracts; the substrate affinities of purified enzymes (K M ) and soil extracts were also similar. The laccase assays showed specificity overlap with tyrosinase and ligninolytic peroxidases when hydrogen peroxide is present. The ABTS oxidation assay is able to reliably detect the presence of 13.5 pg mL À1 or 0.199 Â 10 À12 mol mL À1 of T. versicolor laccase, which is three times more sensitive than the 2,6-dimethoxyphenol-based assay and more than 40 times more sensitive than any of the other assays. The low molecular mass soil-derived compounds and the isolated fulvic and humic acids influence the laccase assays and should be removed from the soil extracts before measurements of the enzyme activity are performed.

Biology and Fertility of Soils, 2010

The small-scale distribution of activities of extracellular laccase, Mn-peroxidase, endoglucanase... more The small-scale distribution of activities of extracellular laccase, Mn-peroxidase, endoglucanase, cellobiohydrolase, β-glucosidase, endoxylanase, βxylosidase, chitinase, and acid phosphatase were studied in the litter (L) and organic (H) horizons of Quercus petraea forest soil and related to the distribution of microbial biomass. Geostatistical analysis showed that the spatial autocorrelation of the enzyme activities and soil microbial biomass measured as phospholipid fatty acids (PLFA) and ergosterol content occurred at similar scales, typically in the range of tens of centimeters. The size of the spatial structures differed between the L and H horizons; for most of the studied enzymatic processes, litter exhibited a higher spatial variability (smaller autocorrelation distances). The distribution of several enzymes, including laccase, Mn-peroxidase, and some hydrolases, reflected the distribution of fungal biomass. Polysaccharide hydrolases exhibited similar spatial distribution patterns in the L horizon, and their activity coincided with a high fungal/ bacterial biomass ratio.

Biodegradation, 2011

Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cell... more Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cellulose in dead wood and as such they attracted considerable attention. The aims of this work were to quantify the activity and spatial distribution of extracellular enzymes in coarse wood colonised by the white-rot basidiomycete Fomes fomentarius and in adjacent fruitbodies of the fungus and to analyse the diversity of the fungal and bacterial community in a fungus-colonised wood and its potential effect on enzyme production by F. fomentarius. Fungus-colonised wood and fruitbodies were collected in low management intensity forests in the Czech Republic. There were significant differences in enzyme production by F. fomentarius between Betula pendula and Fagus sylvatica wood, the activity of cellulose and xylan-degrading enzymes was significantly higher in beech wood than in birch wood. Spatial analysis of a sample B. pendula log segment proved that F. fomentarius was the single fungal representative found in the log. There was a high level of spatial variability in the amount of fungal biomass detected, but no effects on enzyme activities were observed. Samples from the fruiting body showed high b-glucosidase and chitinase activities compared to wood samples. Significantly higher levels of xylanase and cellobiohydrolase were found in samples located near the fruitbody (proximal), and higher laccase and Mn-peroxidase activities were found in the distal ones. The microbial community in wood was dominated by the fungus (fungal to bacterial DNA ratio of 62-111). Bacterial abundance composition was lower in proximal than distal parts of wood by a factor of 24. These results show a significant level of spatial heterogeneity in coarse wood. One of the explanations may be the successive colonization of wood by the fungus: due to differential enzyme production, the rates of biodegradation of coarse wood are also spatially inhomogeneous.

Applied Soil Ecology, 2010

The ISME Journal, 2012

Soils of coniferous forest ecosystems are important for the global carbon cycle, and the identifi... more Soils of coniferous forest ecosystems are important for the global carbon cycle, and the identification of active microbial decomposers is essential for understanding organic matter transformation in these ecosystems. By the independent analysis of DNA and RNA, whole communities of bacteria and fungi and its active members were compared in topsoil of a Picea abies forest during a period of organic matter decomposition. Fungi quantitatively dominate the microbial community in the litter horizon, while the organic horizon shows comparable amount of fungal and bacterial biomasses. Active microbial populations obtained by RNA analysis exhibit similar diversity as DNA-derived populations, but significantly differ in the composition of microbial taxa. Several highly active taxa, especially fungal ones, show low abundance or even absence in the DNA pool. Bacteria and especially fungi are often distinctly associated with a particular soil horizon. Fungal communities are less even than bacterial ones and show higher relative abundances of dominant species. While dominant bacterial species are distributed across the studied ecosystem, distribution of dominant fungi is often spatially restricted as they are only recovered at some locations. The sequences of cbhI gene encoding for cellobiohydrolase (exocellulase), an essential enzyme for cellulose decomposition, were compared in soil metagenome and metatranscriptome and assigned to their producers. Litter horizon exhibits higher diversity and higher proportion of expressed sequences than organic horizon. Cellulose decomposition is mediated by highly diverse fungal populations largely distinct between soil horizons. The results indicate that low-abundance species make an important contribution to decomposition processes in soils.