Stéven Criquet

Annual variations of phenoloxidase activities in an evergreen oak litter: influence of certain biotic and abiotic factors.

This study concerns ligninolysis phenomena occurring over 13 months in forest litter. Evergreen oak (Quercus ilex L.) litter was taken as a model because Quercus ilex L. is the most abundant tree species in forests of the French Mediterranean area. Several biotic and abiotic factors potentially involved in transformations of polyphenolic compounds, were measured between October 1997 and October 1998. These factors were: global fungal microflora, the fungi producing phenoloxidases (PO + ), the activities of several phenoloxidases, hydrosoluble phenols, and temperature, humidity and pH of the litter. Results showed that the annual dynamics of fungi and phenoloxidase activities appear to be seasonal, i.e. that these biotic factors such as, were optimal in autumn. A multiple regression analysis showed that there was no correlation between biotic factors such as, fungal populations and phenoloxidase activity and abiotic factors such as, temperature, humidity and pH. Laccases were the preponderant phenoloxidase activities during the year, while those of Mn-peroxidases only appeared in the autumn of 1997. Other phenoloxidases, lignin-peroxidases and tyrosinases were never detected. Interactions between laccases and humic substances were also investigated. Adsorption of laccases on humic substances leads to a shift in the optimal temperature activity of these enzymes from 50 to 308C. Activities of laccases also shifted towards more acidic values when laccases were not adsorbed on humic substances. Nevertheless, the optimal pH was the same (5.7) whether laccases were adsorbed or not to humic substances. Electrophoresis analysis showed little variations in the number of phenoloxidase isoenzymes. Indeed, laccases showed three isoenzymes during the year (Rf 0.23, 0.34 and 0.43). Only one isoform of Mn-peroxidase, with an Rf 0.21, was detected in the litter. 7 2000 Elsevier Science Ltd. All rights reserved.

Laccase activity of forest litter

Abstract Laccases are blue-copper enzymatic proteins involved in the transformation of polyphenolic compounds, such as lignin, into forest litter. A method to measure their activity was developed using an evergreen oak litter as a model system. We considered factors that affect the extraction of these enzymes and the measurement of their activity. The factorsstudied included powdering of the leaves, adding polyvinylpolypyrrolidone or Tween 80, adsorption of enzymes on centrifugation pellets, concentration of the extracts, nature and molarity of the extraction solvent, amount of powdered leaves used for enzymatic extraction, extraction time, pH, and temperature during the measurement of the activity.

Endoglucanase and β-glycosidase activities in an evergreen oak litter: annual variation and regulating factors

Abstract This paper represents the third part of a larger study about the processes involved in litter biodegradation in an oak copse in France. It concerns the variations in activities of endoglucanases and of β-glycosidases over 13 months in forest litter. Evergreen oak ( Quercus ilex L.) litter was taken as a model because Q. ilex is the most abundant tree species in forests of the French Mediterranean area. The activities of several endoglucanases and β-glycosidases, and the concentrations of the proteins and reducing sugars were measured in the evergreen oak litter between October 1997 and October 1998. Several biotic and abiotic factors, potentially involved in the regulation of the activities of these hydrolyzing enzymes, were also measured: culturable heterotrophic bacteria, cellulolytic bacteria and cellulolytic fungi, moisture, temperature and pH of the litter. Under this Mediterranean climate, the moisture of the litter was strongly correlated with many factors and is likely to be important in regulating the temporal dynamics of various biological parameters. A multiple regression analysis showed that the annual dynamic of the culturable heterotrophic bacteria was primarily influenced by moisture but also by variations in concentration of reducing sugars in the litter. Moreover, these bacteria were correlated with the activities of several β-glycosidases, and specifically with the N -acetyl-glucosaminidases ( r =0.93). No relationships were established between the number of cellulolytic microorganisms (bacteria and fungi) and the activities of endoglucanases (i.e. cellulases and hemicellulases). The seasonal trends of certain biotic processes, and the influence of pH and of temperature on these processes are also discussed. Electrophoretic analyses were used to estimate the number of cellulase and xylanase isoenzymes in the litter, and to study their fluctuations over one year. The prevailing cellulase isoenzyme had a R f of 0.64, and there was no relationship between the abundance of cellulolytic microorganisms and the electrophoretic profiles of the cellulases. In the case of the xylanases, the electrophoretic profiles were homogenous throughout the year, and the prevailing xylanase had a R f of 0.75.

Measurement and characterization of cellulase activity in sclerophyllous forest litter

Cellulases are enzymatic proteins which hydrolyze cellulose polymers to smaller oligosaccharides, cellobiose and glucose. They consist in three major types of enzymes: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and beta-glucosidases (EC 3.2.1.21) which play an essential role in carbon turnover of forest ecosystem. The aim of this study was firstly to determine the parameters (i.e. buffer type, pH, temperature, quantity of litter, incubation time and reagent type) which affect the measurement of cellulase activity in a sclerophyllous forest litter, and secondly to compare two methods for measuring cellulase activity: a direct method and an extraction method. In the direct method, the litter was directly incubated with a buffered solution containing the enzyme substrate, whereas in the extraction method, the cellulases were firstly extracted before measuring their activity. The results were compared with other studies about soil cellulase activity, and it appeared that several parameters (buffer type, pH, temperature and sample quantity) which influence the measurement of cellulase activity differ according to whether a soil or a litter is considered. Concerning the procedure used for the measurement of cellulase activity, results showed that the activity values were higher when using an extraction procedure than when using a direct procedure. The extraction procedure, combined with a concentration stage of the extract, also allowed electrophoretic analysis (PAGE) of the cellulases extracted from the litter. The electrophoretic pattern revealed two cellulase isoenzymes which may be related to the occurrence of two pH-activity peaks of these enzymes when citrate buffer was used for the measurement of cellulase activity in the litter.

Biological functioning of PAH-polluted and thermal desorption-treated soils assessed by fauna and microbial bioindicators

A large number of soil bioindicators were used to assess biological diversity and activity in soil polluted with polycyclic aromatic hydrocarbons (PAHs) and the same soil after thermal desorption (TD) treatment. Abundance and biodiversity of bacteria, fungi, protozoa, nematodes and microarthropods, as well as functional parameters such as enzymatic activities and soil respiration, were assessed during a two year period of in situ monitoring. We investigated the influence of vegetation (spontaneous vegetation and Medicago sativa) and TD treatment on biological functioning. Multivariate analysis was performed to analyze the whole data set. A principal response curve (PRC) technique was used to evaluate the different treatments (various vegetation and contaminated vs. TD soil) contrasted with control (bare) soil over time. Our results indicated the value of using a number of complementary bioindicators, describing both diversity and functions, to assess the influence of vegetation on soil and discriminate polluted from thermal desorption (TD)-treated soil. Plants had an influence on the abundance and activity of all organisms examined in our study, favoring the whole trophic chain development. However, although TD-treated soil had a high abundance and diversity of microorganisms and fauna, enzymatic activities were weak because of the strong physical and chemical modifications of this soil.

2,7-Diaminofluorene is a sensitive substrate for detection and characterization of plant root peroxidase activities

The 2,7-diaminofluorene (DAF) was a far more sensitive substrate for lucerne root peroxidases than were guaiacol or pyrogallol. All the peroxidase assays had an optimum pH of 6. The optimum temperature for the activity of DAF-peroxidase was 35 °C, far lower than those of guaiacol- and pyrogallol-peroxidases (60 °C). Electrophoresis of lucerne root peroxidases revealed the presence of eight isoenzymes using DAF as substrate, and only two with guaiacol. The influence of four organic solvents on peroxidase activity was also assessed.

Protein measurement in forest litter

Abstract. A method to extract proteins and measure their concentrations in forest litter was developed using evergreen oak litter as a model system. We considered factors that affect the extraction of proteins and the measurement of their concentrations. The factors studied included powdering of the leaves, nature and molarity of the extraction solvent, amount of powdered leaves used for protein extraction, concentration of the extracts, adding polyvinylpolypyrrolidone (PVPP) and Tween 80 during the extraction, extraction time, incubation time with the colorimetric reagent and colorimetric method used. Results showed in particular that CaCl2 was the only extraction solvent which allowed one to obtain extracts without interfering substances for the measurement of protein concentrations. Adding PVPP and Tween 80 during the extraction procedure led to an increase in the protein concentrations in extracts by eliminating precipitating substances such as tannins. The modalities of the other parameters were also optimised and are discussed for more accurate protein measurement.

Functional Patterns of Microbial Communities of Rhizospheric Soils Across the Development Stages of a Young Mangrove in French Guiana

The functional patterns of microbial communities (microbial respiration, enzyme activities, functional diversity) and the relevant physico-chemical characteristics of rhizospheric soils were studied during the process of mudflat colonization by mangrove. The study site is a fringe mangrove stand located in Montabo Bay at Cayenne (French Guiana). It is characterized by different vegetation development stages dominated by an assemblage of Avicennia germinans and Laguncularia racemosa. Rhizospheric and surface soils were collected from three stations based on successional stages of mangrove colonization: pioneer (P), coppice (C), and young forest (F). The microbial functional patterns showed significant progressive shifts along the mangrove vegetation profile. The P stages, those most influenced by tide currents, were macroscopically characterized by hydro-sedimentary instability and micro-phytobenthic colonization of mudflat. This stage, characterized by low total organic carbon (TOC) content and quality, showed the lowest extracellular enzymatic activities and the highest functional metabolic diversities. TOC quality analyses by 13C CPMAS NMR provided evidence of progressive TOC enrichment and an increasing imprint of aboveground vegetation on C quality as succession occurs. These differences in the origin, amount, and quality of soil organic matter (SOM) of older stages exerted both a quantitative and qualitative control over microbial functional responses. This indicated the enhancement of aboveground–belowground functional linkages, leading to the expression of high decomposition activities and a functional loss and specialization of rhizospheric microbial communities.