Degradation of Humic Acids by the Litter-Decomposing Basidiomycete Collybia dryophila

The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic ¹⁴C-labeled humic acid (¹⁴C-HA) prepared from [U-¹⁴C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn²⁺ (200 μM), leading to 75% conversion of LHA and 50% mineralization of ¹⁴C-HA (compared to 60% and 20%, respectively, in the absence of Mn²⁺). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn²⁺-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.     

Degradation of humic acids by the litter-decomposing basidiomycete Collybia dryophila

The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic (14)C-labeled humic acid ((14)C-HA) prepared from [U-(14)C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn(2+) (200 microM), leading to 75% conversion of LHA and 50% mineralization of (14)C-HA (compared to 60% and 20%, respectively, in the absence of Mn(2+)). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn(2+)-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.     

Endoglucanase Activity of Compost-Dwelling Fungus Paecilomyces inflatus is Stimulated by Humic Acids and Other Low Molecular Mass Aromatics

Summary Paecilomyces inflatus isolated from municipal waste compost was found to have cellulolytic activity in several solid and liquid media. This study was done to reveal the multifarious effects of municipal waste compost on endoglucanase activity of P. inflatus. The highest enzyme activities under the conditions of solid-state fermentation were measured in authentic compost samples compared with wood, straw and bran substrates. In surface liquid cultures glucose, cellobiose, xylan, Avicel cellulose, carboxymethylcellulose (CM-cellulose), starch and citrus pectin were used as carbon sources. All carbon sources supported the growth of P. inflatus. However, only CM-cellulose, cellobiose and pectin noticeably stimulated endoglucanase (EG) activity. Further stimulation of EG activity was obtained in cultures containing 1% CM-cellulose as a carbon source by supplementation with low-molecular mass aromatic compounds vanillin, veratric acid and benzoic acid, and with soil humic acid (SHA). SHA and veratric acid were found to be the most efficient elicitors of the cellulolytic activity. P. inflatus was able to utilize nitrate and ammonium as pure nitrogen sources in media containing cellulose.     

UPTAKE OF LIPOPHILIC CADMIUM COMPLEXES BY THREE GREEN ALGAE: INFLUENCE OF HUMIC ACID AND ITS pH DEPENDENCE1

Cadmium forms neutral, lipophilic CdL₂⁰ complexes with diethyldithiocarbamate (L = DDC) and with ethylxanthate (L = XANT). In a synthetic solution and in the absence of natural dissolved organic matter (DOM), for a given total Cd concentration, uptake of these complexes by unicellular algae is much faster than the uptake of the free Cd²⁺ cation. The objective of the present study was to determine how this enhanced uptake of the lipophilic CdL₂⁰ complexes was affected by the presence of natural DOM (Suwannee River humic acid, SRHA). Experiments were performed with Cd(DDC)₂⁰ and Cd(XANT)₂⁰ at two pH values (7.0 and 5.5) and with the three chlorophytes [Chlamydomonas reinhardtii P. A. Dang., Pseudokirchneriella subcapitata (Korshikov) Hindák, Chlorella fusca var. vacuolata Shihira et R. W. Krauss]. Short‐term uptake (30–40 min) of the CdL₂⁰ complexes was followed in the absence and presence of SRHA (6.5 mg C · L^?1). Acidification from pH 7.0 to 5.5 decreased CdL₂⁰ uptake by the three algae, in the presence or absence of humic acid (HA). The dominant effect of the HA was to decrease Cd uptake, due to its interaction with the CdL₂⁰ complexes in solution. However, if uptake of the free CdL₂⁰ complexes was compared in the presence and absence of HA, in four of eight cases initial uptake rate constants (k_i) were significantly higher (P < 0.05) in the presence of the HA, suggesting the operation of an interfacial effect of the HA at the algal cell membrane, favoring uptake of CdL₂⁰. Overall, the experimental results suggest that neutral metal complexes will be less bioavailable in natural waters than they are in synthetic laboratory media in the absence of natural DOM.     

Degradation and enzymatic activities of three Paecilomyces inflatus strains grown on diverse lignocellulosic substrates

This paper describes for the first time in detail the lignocellulose degradation system in Paecilomyces inflatus. The fungal genus Paecilomyces contributes the carbon turnover from lignin and carbohydrate plant residues, particularly in compost and soil environment, where basidiomycetes appear very seldom. We studied three different strains of P. inflatus, obtained from different ecophysiological and geographical origin. Various cultivation conditions were employed, and the chemical analysis of decayed straw, compost, birch and spruce wood chips indicated variable responses. Endoglucanase, xylanase and laccase were assayed. All strains of P. inflatus, regardless of their origin, altered the ambient pH in a similar manner in all investigated substrates, suggesting that all P. inflatus isolates may share the common regulatory system to control their environmental pH. The variability among strains of P. inflatus in their ability to remove lignocellulose components often was related to the nature of the substrate and the production of specific enzymes although it was not strictly correlated. This may implicate that other enzymes and/or even other parameters needed for lignocellulosics degradation in P. inflatus should be evaluated. Indications for specific adaptation strategies that may operate in P. inflatus were found.     

Hyaluronate-cell interaction : Effects of exogenous hyaluronate on muscle fibroblast cell surface composition

Evidence that exogenous hyaluronate (HA) binds to the surface of muscle fibroblast cultures was obtained by incubating confluent fibroblasts with ¹⁴C-HA purified from fibroblast cell surfaces. Surface-bound ¹⁴C-HA was operationally defined as material resistant to six saline washes and solubilized by brief trypsinization. All of the surface-bound radioactivity remains as authentic HA. Exposure of fibroblasts to 100 μg/ml cold HA caused a nearly 3-fold ‘reduction’ in incorporation of isotopic precursors into glycosaminoglycan (GAG); but when intracellular ¹⁴C precursors to GAG were quantitated, the entire ‘reduction’ could be accounted for by decreased precursor uptake. Exposure to exogenous HA altered the distribution of newly synthesized GAG by stimulating an increase in total GAG secreted to the medium at the expense of that bound to the culture surface. Qualitatively, the cell surface ratio of ¹⁴C-HA: ¹⁴C-sulfated GAG (SGAG) of HA-treated cells is about 2.5 times greater than that of untreated cells and the medium ratio is correspondingly reversed. This is primarily the result of stimulated ¹⁴C-SGAG release to the culture medium. Addition of cold HA to prelabeled cultures also stimulates the selective turnover of SGAG from the culture surface. Thus, exposure to HA alters the fibroblast surface by accumulation of exogenous HA as well as by stimulation of SGAG turnover.     

Microbial Decomposition of Synthetic 14C-Labeled Lignins in Nature: Lignin Biodegradation in a Variety of Natural Materials

Lignin biodegradation in a variety of natural materials was examined using specifically labeled synthetic ¹⁴C-lignins. Natural materials included soils, sediments, silage, steer bedding, and rumen contents. Both aerobic and anaerobic incubations were used. No ¹⁴C-labeled lignin biodegradation to labeled gaseous products under anaerobic conditions was observed. Aerobic ¹⁴C-labeled lignin mineralization varied with respect to type of natural material used, site, soil type and horizon, and temperature. The greatest observed degradation occurred in a soil from Yellowstone National Park and amounted to over 42% conversion of total radioactivity to ¹⁴CO₂ during 78 days of incubation. Amounts of ¹⁴C-labeled lignin mineralization in Wisconsin soils and sediments were significantly correlated with organic carbon, organic nitrogen, nitrate nitrogen, exchangeable calcium, and exchangeable potassium.     

Degradation of softwood [14C lignin] lignocelluloses and its relation to the formation of humic substances in river and pond environments

The fate of lignin in water and sediment of the Garonne river (France) and of a pond in its floodplain was examined using specifically labeled [¹⁴C-lignin] lignocelluloses. No significant differences appeared in the mineralization rate of alder, poplar or willow [¹⁴C-lignin] in running water samples. Conversion of total radioactivity to ¹⁴CO₂ ranged between 18.7% and 24.4% after 120 days of incubation. Degree of ¹⁴C-labeled lignin mineralization in standing water and sediments was clearly lower, especially in submerged sediments, and was correlated with oxygen supply. After 60 days of incubation 3.3% to 7.9% of the ¹⁴C-labeled lignin was recovered in water samples as dissolved organic carbon originating from microbial metabolism. In water extracts from sediment the percentage of dissolved organic ¹⁴C was only 0.4% to 1.3% of the applied activity. In the humic fraction extracted from sediments it did not exceed 4.4% which was much lower than in soils. No significant difference appeared between river and pond conditions for humic substances formation.     

Evaluation of the transformation of organic matter to humic substances in compost by coupling sec-page

Humic acids (HAs) from soil and compost at the beginning (S0) and at the end of the stabilization process after 130 days (S130) have been fractionated by coupling size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). Preparative quantities of HA fractions (HAFs) with different molecular sizes (MSs) and exactly defined electrophoretic mobility (EMs) have been obtained from all samples and the HAFs weight content has been studied. A high degree of similarity in HAFs weight content between soil HA and a stabilized compost HAs130 has been observed. Such data seem to be reliable for monitoring the evolution of the compost organic matter to humic substances for their agricultural uses.     

Structural Alteration of Humic Acids by Pseudomonas spp. from Deep Terrestrial Subsurface Diatomite Formations in Northernmost Japan

Bacterial utilization of humic acids (HAs) was examined under aerobic conditions using Pseudomonas spp. from diatomite from a depth of 250 m below ground level in the Horonobe Underground Research Laboratory. HA decolorization and bacterial aggregation were observed during cultivation when an auxiliary carbon source was added. High-performance size-exclusion chromatography showed that high-molecular-weight HAs were produced. Fourier transform infrared spectroscopy indicated that carboxyl groups and polysaccharide-related substances in HAs were eliminated, while aliphatic structural units and amide groups were added to HAs. These results suggested that Pseudomonas spp. utilize and alter the molecular structure of HAs under aerobic conditions caused by the construction of underground facilities.     

Comparative Sorption of Phenanthrene and Benzo[α]pyrene to Soil Humic Acids

Sorption of phenanthrene (Phen) and benzo[α]pyrene (BaP) by humic acids (HAs) extracted from four typical soils in China, including cinnamon soil, fluvo-aquic soil, red soil, and mountain meadow soil were investigated. All sorption data were fitted well by the Freundlich model, but BaP exhibited stronger and more nonlinear sorption than Phen by a given HA. For Phen isotherms, there was a positive relation between K _oc values and aliphaticity of HAs, whereas a negative correlation was observed between n values and aliphaticity. This indicated the importance of aliphatic groups in Phen sorption capacity and nonlinearity. Compared to Phen, a similar trend was obtained between n values of BaP and aliphaticity or aromatic carbon. However, no correlation existed between K _oc values for BaP and aliphaticity or aromatic carbon.     

Biodegradation of 14C-Labeled Model and Cornstalk Lignins, Phenols, Model Phenolase Humic Polymers, and Fungal Melanins as Influenced by a Readily Available Carbon Source and Soil

After 6 months of incubation in a fertile neutral sandy loam, about 48% of the ring carbons and 2-carbons and 60% of the OCH₃ carbons of specifically labeled coniferyl alcohol had evolved as CO₂. After 1 year, corresponding values were 55 and 65%. When coniferyl alcohol units were linked into model and cornstalk lignins, about 23% of the ring carbons and 2-carbons and 39% of the OCH₃ carbons had evolved as CO₂ after 6 months. After 1 year, corresponding values were about 28 and 46%. The addition of orange leaves (0.5%, wt/wt) after 6 months did not significantly increase the evolution of ¹⁴CO₂. Addition of orange leaves (0.5%, wt/wt) with specifically ¹⁴C-labeled pyrocatechol, coumaryl alcohol, model lignins, humic acid-type phenolic polymers and of uniformly ¹⁴C-labeled fungal melanins did not increase labeled C losses or C losses from the orange leaves. Decomposition of protein and pyrocatechol linked into model humic acid polymers, coniferyl alcohol C in model lignins, and Eurotium echinulatum melanin in six soils varied from 2 to 14%. Significant differences in C losses were related to soils and were not influenced by orange leaf applications.     

Earthworms as modifiers of the structure and biological activity of humic acids

Passage of humic acids (HAs) through the digestive tract of the earthworm, Eisenia fetida andrei, resulted in a decrease in molecular masses of the HAs. The effect of earthworm-modified HAs on individual bacteria and on bacterial communities as a whole is different from the effect of native HAs. Modified HA probably induces and regulates microbial successions in soils and composts in a different manner than the native HA, suppressing or stimulating different groups of microorganisms. These results may explain why the positive effects of commercial humates in real soil ecosystems, unlike model communities, attenuate rapidly.     

Degradation of lignite (low-rank coal) by ligninolytic basidiomycetes and their manganese peroxidase system

Ligninolytic basidiomycetes (wood and leaf-litter-decaying fungi) have the ability to degrade low-rank coal (lignite). Extracellular manganese peroxidase is the crucial enzyme in the depolymerization process of both coal-derived humic substances and native coal. The depolymerization of coal by Mn peroxidase is catalysed via chelated Mn(III) acting as a diffusible mediator with a high redox potential and can be enhanced in the presence of additional mediating agents (e.g. glutathione). The depolymerization process results in the formation of a complex mixture of lower-molecular-mass fulvic-acid-like compounds. Experiments using a synthetic ¹⁴C-labeled humic acid demonstrated that the Mn peroxidase-catalyzed depolymerization of humic substances was accompanied by a substantial release of carbon dioxide (17%–50% of the initially added radioactivity was released as ¹⁴CO₂). Mn peroxidase was found to be a highly stable enzyme that remained active for several weeks under reaction conditions in a liquid reaction mixture and even persisted in sterile and native soil from an opencast mining area for some days. Received: 31 July 1998 / Received revision: 29 September 1998 / Accepted: 2 October 1998     

Humic acids isolated from earthworm compost enhance root elongation,lateral root emergence,and plasma membrane H+-ATPase activity in maize roots

Earthworms (Eisenia foetida) produce humic substances that can influence plant growth by mechanisms that are not yet clear. In this work, we investigated the effects of humic acids (HAs) isolated from cattle manure earthworm compost on the earliest stages of lateral root development and on the plasma membrane H(+)-ATPase activity. These HAs enhance the root growth of maize (Zea mays) seedlings in conjunction with a marked proliferation of sites of lateral root emergence. They also stimulate the plasma membrane H(+)-ATPase activity, apparently associated with an ability to promote expression of this enzyme. In addition, structural analysis reveals the presence of exchangeable auxin groups in the macrostructure of the earthworm compost HA. These results may shed light on the hormonal activity that has been postulated for these humic substances.     

Involvement of ligninolytic enzymes and Fenton-like reaction in humic acid degradation by Trametes sp

Trametes sp. M23, isolated from biosolids compost was found to decompose humic acids (HA). A low N (LN) medium (C/N, 53) provided suitable conditions for HA degradation, whereas in a high N (HN) medium (C/N, 10), HA was not degraded. In the absence of Mn²⁺, HA degradation was similar to that in Mn²⁺-containing medium. In contrast, MnP activity was significantly affected by Mn²⁺. Laccase activity exhibited a negative correlation to HA degradation, while LiP activity was not detected. Thus, ligninolytic enzymes activity could provide only a partial explanation for the HA-degradation mechanism. The decolorization of two dyes, Orange II and Brilliant Blue R250, was also determined. Similar to HA degradation, under LN conditions, decolorization occurred independently of the presence of Mn²⁺. We investigated the possible involvement of a Fenton-like reaction in HA degradation. The addition of DMSO, an OH-radical scavenger, to LN media resulted in a significant decrease in HA bleaching. The rate of extracellular Fe³⁺ reduction was much higher in the LN vs. HN medium. In addition, the rate of reduction was even higher in the presence of HA in the medium. In vitro HA bleaching in non-inoculated media was observed with H₂O₂ amendment to a final concentration of 200 mM (obtained by 50 mM amendments for 4 days) and Fe²⁺ (36 mM). After 4 days of incubation, HA decolorization was similar to the biological treatment. These results support our hypothesis that a Fenton-like reaction is involved in HA degradation by Trametes sp. M23.     

Humic acid differentially improves nitrate kinetics under low‐ and high‐affinity systems and alters the expression of plasma membrane H+‐ATPases and nitrate transporters in rice

Humic acids (HAs) have a major effect on nutrient uptake, metabolism, growth and development in plants. Here, we evaluated the effect of HA pretreatment applied with a nutrient solution on the uptake kinetics of nitrate nitrogen (N‐NO₃^?) and the metabolism of nitrogen (N) in rice under conditions of high and low NO₃^? supply. In addition, the kinetic parameters of NO₃^? uptake, N metabolites, and nitrate transporters (NRTs) and the plasma membrane (PM) H⁺‐ATPase gene expression were examined. The plants were grown in a growth chamber with modified Hoagland and Arnon solution until 21 days after germination (DAG), and they were then transferred to a solution without N for 48 h and then to another solution without N and with and without the addition of HAs for another 48 h. After this period of N deprivation, the plants received new nutrient solutions containing 0.2 and 2.0 mM N‐NO₃^?. Treatment of rice plants with HA promoted the induction of the genes OsNRT2.1‐2.2/OsNAR2.1 and some isoforms PM H⁺‐ATPase in roots. The application of HAs differentially modified the parameters of the uptake kinetics of NO₃^? under both concentrations. When grown with 0.2 mM NO₃^?, the plants pretreated with HA had lower K_m and C_min values as well as a higher V_max/K_m ratio. When grown with 2 mM NO₃^?, the plants pretreated with HA had a higher V_max value, a greater root and shoot mass, and a lower root/shoot ratio. The N fractions were also altered by pretreatment with HA, and a greater accumulation of NO₃^? and N‐amino was observed in the roots and shoots, respectively, of plants pretreated with HA. The results suggest that pretreatment with HA modifies root morphology and gene expression of PM H⁺‐ATPases and NO₃^? transporters, resulting in a greater efficiency of NO₃^? acquisition by high‐ and low‐affinity systems.     

Mineralization of synthetic humic substances by manganese peroxidase from the white-rot fungus Nematoloma frowardii

A manganese peroxidase preparation from the white-rot fungus Nematoloma frowardii was found to be capable of releasing up to 17% ¹⁴CO₂ from ¹⁴C-labelled synthetic humic substances. The latter were prepared from [U-¹⁴C]catechol by spontaneous oxidative polymerization or laccase-catalysed polymerization. The ex-tent of humic substance mineralization was considerably enhanced in the presence of the thiol mediator glutathione (up to 50%). Besides the evolution of ¹⁴CO₂, the treatment of humic substances with Mn peroxidase resulted in the formation of lower-molecular-mass products. Analysis of residual radioactivity by gel-permeation chromatography demonstrated that the predominant molecular masses of the initial humic substances ranged between 2 kDa and 6 kDa; after treatment with Mn peroxidase, they were reduced to 0.5–2 kDa. The extracellular depolymerization and mineralization of humic substances by the Mn peroxidase system may play an important role in humus turnover of habitats that are rich in basidiomycetous fungi. Received: 25 September 1997 / Received revision: 12 January 1998 / Accepted: 13 January 1998     

Photochemical mineralization of synthetic lignin in lake water indicates enhanced turnover of aromatic organic matter under solar radiation

The degradation of ¹⁴C-[ring]-labelled syntheticlignin (¹⁴C-DHP) and dissolved organic carbon(DOC) from lake water were studied simultaneously.¹⁴C-DHP was incubated in humic lake water (colour173 mg Pt l^-1) for 7 d in the dark or under solarradiation. In the dark <0.4% of the introduced¹⁴C-DHP label and 4% of the indigenous DOC weremineralized, indicating that the ¹⁴C-labelledaromatic rings of DHP and the humic DOC weremicrobiologically recalcitrant. Under solar radiation(116 MJ m^-2), 17–21% of the ¹⁴C-labelledcarbons in DHP and 18–23% of the indigenous DOC weremineralized in 7 d. Simultaneously the watersolubility of ¹⁴C-DHP increased. Solar radiationconverted the aromatic cores of synthetic lignin toCO₂ and soluble organic photoproducts. Theresults suggest that solar radiation plays a key rolein the decomposition of natural polyaromatic matter.     

Fate of 14C-labeled anthracene and hexadecane in compost-manured soil

Experiments were carried out to evaluate the impact of the addition of ripe compost on the degradation of two ¹⁴C-labeled hydrocarbon model compounds (anthracene and hexadecane) in soil. The addition of mature compost (20 % dry wt./dry wt.) stimulated significantly the disappearance of the extractable fraction of both compounds. With compost, 23 % of the labeled anthracene was transformed into ¹⁴CO₂ and 42 % was fixed to the soil matrix irreversibly. In the unsupplemented control reactor more than 88 % of the original anthracene could be recovered by either of two applied organic extraction procedures. The formation of non-extractable bound residues was less significant with [¹⁴C] hexadecane since only 21 % of the labeled carbon had become non-extractable after 103 days. The results presented show that compost could stimulate the depletion of hydrocarbons by either mineralization or the formation of unextractable bound residues (humification). The latter process might be a significant route of depletion in soil especially, for those hydrocarbons that are mineralized only slowly. The meaning of this finding for the assessment of soil bioremediation is discussed.These authors contributed equally to the presented work and should therefore both be considered as first authors