Biodegradation of humic substances by microscopic filamentous fungi: chromatographic and spectroscopic proxies

Purpose

The study of interactions between humic substances (HSs) and soil filamentous fungi is the key to understanding the sustainable soil functioning. The present work aims to examine the decomposition of HSs by filamentous dark-pigmented fungus Alternaria alternatа under the laboratory conditions and to determine the effect of easily assimilable organic carbon on this process. Analyzing such polydisperse substances like HSs by a complex integrated methodology makes it possible to explore the data on their decomposition by microorganisms.

Materials and methods

To achieve the aforementioned goals, we used chromatographic and spectroscopic approaches: low-pressure size-exclusion and hydrophobic interaction chromatography accompanied by absorption and fluorescence spectroscopy. To determine the effect cometabolism conditions produced on HS decomposition, two types of carbon substrates were added to the nutrient media: easily assimilable organic carbon (standard 0.3% or reduced 0.03% sucrose content) and hardly assimilable organic carbon (HSs), as well as their combinations. Five HS samples of different organic matter origin have been inspected: potassium humates (HPs) and humic acids (HAs) from coal, peat, and lignosulfonate. Correlation matrix and principal component analysis (PCA) were calculated for comprehensive data analysis.

Results and discussion

Transformations of the investigated HSs under fungal cultivation lead to the increase in the low molecular weight fraction, rise of hydrophilic fraction, enlargement of absorbance ratio A₂₅₀/A₃₆₅, shortening of the emission wavelength of the humic-type fluorescence, and growth in the fluorescence quantum yield measured with excitation at 355 nm. A positive correlation was observed between the accumulation of fungal biomass and the degree of HS decomposition. PCA analysis confirms that the difference in the results of HS decomposition largely depends on the sucrose content and the nature of HSs. We divided all the HS samples into four groups according to the degree of HS decomposition: original HS solutions, HPs altered using fungal cultivation at 0.03% sucrose, HAs after fungal cultivation at 0.03% sucrose, and finally, HSs (both HPs and HAs) after fungal cultivation at 0.3% sucrose.

Conclusions

In the laboratory experiments, we showed that (1) the isolated HAs were more effectively degraded than the parent HPs, and this process was more pronounced at a reduced sucrose content, and (2) the decomposition of stable organic compounds (HSs) was activated by the easily assimilable carbon sources (especially 0.3% sucrose) being present. We assume that it is the easily assimilable organic carbon that most likely triggers the HS degradation working as the priming effect in natural environments.

     

Beta-carotene isomer composition of sub- and supercritical carbon dioxide extracts. Antioxidant activity measurement

In the present work sub- and supercritical extraction conditions using carbon dioxide were studied in order to obtain extracts with different compositions from the green microalgae Dunaliella salina. Different compositions of beta-carotene isomers were identified in the extracts by using HPLC-DAD. Also, antioxidant activity of the extracts was measured using a TEAC assay. An experimental design was applied considering two factors, extraction pressure and temperature, in a wide range of values, trying to maximize the extraction yield. Higher yields were obtained at high pressures and low temperatures, that is, at higher CO2 densities. Attempts were made to correlate the antioxidant activity of the extracts with their chemical composition by means of principal component analysis. A certain relationship was found between their antioxidant activity and the isomeric composition of beta-carotenes. As a result, an original equation is proposed to predict the antioxidant activity of extracts from D. salina in terms of the ratio 9-cis-beta-carotene/all-trans-beta-carotene, the concentration of alpha-carotene, and, especially, the concentration of 9-cis-beta-carotene.     

One- and two-dimensional electrophoretic identification of IgE-binding polypeptides of Lupinus albus and other legume seeds

The prevalence of food allergies in the world population requires integrated approaches to identify new potential allergens, especially those of plant origin. The aim of this work was the allergen in vitro analysis of Lupinus albus seed proteome, a promising food protein source, and the assessment of IgE cross-reactivities with other more diffused legume species. A combination of one- and two-dimensional gel electrophoresis and immunoblotting analyses with specific IgGs for band identification and lupin-sensitized patients' circulating IgEs for allergenicity studies has been used. Two lupin proteins, namely, conglutin gamma and 11S globulin basic subunits, strongly reacted with all patients' sera. Also, cross-reactivities with the homologous polypeptides of other legume species were observed. Otherwise, no reaction at all was detected with a 2S-type lupin protein. This global electrophoretic approach has allowed the identification of a new potential lupin allergen and confirmed the cross-reactivity among the legume 11S globulin basic subunits.     

Biostimulant activity of two protein hydrolyzates in the growth and nitrogen metabolism of maize seedlings

Two protein hydrolyzate–based fertilizers (PHFs), one from alfalfa (AH) and one from meat flour (MFH), were studied chemically and biologically. AH and MFH revealed a different degree of hydrolysis and a different amino acid composition. The biostimulant activity was investigated using two specific and sensitive bioassays of auxins and gibberellins. Extracts of AH and MFH elicited a gibberellin‐like activity and a weak auxin‐like one. To improve our understanding of the biostimulant activity, AH and MFH were supplied to maize plants and their effect on growth and nitrate metabolism was studied. Both PHFs increased root and leaf growth and induced morphological changes in root architecture. Besides, the treatments increased nitrate reductase (NR) and glutamine synthetase (GS) activities, suggesting a positive role of the two hydrolyzates in the induction of nitrate conversion into organic nitrogen. Moreover, treatments enhanced GS1 and GS2 isoforms in maize leaves. The latter isoform, amounting to 5‐ to 7‐fold the level of the former, appears to be a superior form in the assimilation of ammonia. The high NR and GS activities together with the high induction of GS isoforms indicate a stimulatory effect of the two PHFs on the assimilation of nitrate. In addition, a role of amino acids and small peptides of the two PHFs is suggested in the regulation of the hormone‐like activity and nitrogen pathway.     

Pre-Harvest Rind-Spotting in ‘Clementine’ Mandarin

A severe physiological disorder of ‘Clementine’ mandarins in the Assini area of Argolis, Southestern Greece, was investigated for two successive years. Irregular-shaped necrotic spots were observed on the rinds of mandarins, mainly at the fruit stylar-ends, from the color break till harvest time. In order to investigate the possible correlation between the disorder and the inorganic nutrition of the fruit, spotted and symptomless mandarin rinds, in four commercial orchards were chemically analyzed. The relationship between fruit inorganic nutrition and the disorder symptoms were not consistent. Using Electron Scanning Microscopy, invisible cracks were observed at the cuticle and the external strata of the spotted rind whereas oil glands remained intact. Considering the results of chemical analyses, microscopic observations, as well as, the information given by the growers, this physiological disorder should be attributed to sudden changes of climatic conditions and not to nutritional imbalance.     

Fate of prions in soil: Degradation of recombinant prion in aqueous extracts from soil and casts of two earthworm species

Prions represent the active agent in transmissible spongiform encephalopathy (TSE) diseases and can remain infective to mammals even after prolonged periods in soil. The influence of mesofauna on prion dispersal and degradation in soil, however, remains unknown. In this study the effect of earthworms on the retention/dissemination of TSEs in soil was evaluated using a model recombinant prion protein (recPrP) and aqueous extracts from soil and fresh casts of two earthworm species, Lumbricus terrestris and Aporrectodea caliginosa. Our results showed that earthworm gut-derived enzymes did not enhance the degradation of recPrP in comparison to soil, even though non-prion related proteolytic activity was higher in fresh worm excrements than in soil samples. Complete degradation of recPrP occurred in the aqueous extracts from all samples within up to 6 days at +15 °C. The proteolytic enzymes responsible for degrading recPrP were inhibited by aprotinin and leupeptin and studies in pure cultures suggested these were most probably of soil microbial origin.     

Water mobility in multicomponent model media as studied by 2H and 17O NMR

Molecular mobility of water was studied in a microbiological media containing complex and heterogeneous mixtures of cellulose, l-sorbose, and orange serum broth (OSB) using (2)H and (17)O high-resolution NMR. All mixtures showed Lorentian (17)O NMR spectra but complex (2)H NMR line shapes. Sorbose, when solubilized, caused line-narrowing where as cellulose-OSB mixtures showed wide peaks with flat plateaus. Presence of liquid or solvent water had a profound effect on a marked increase in T(2) relaxation time observed in sorbose-containing samples. (17)O NMR data were not composition dependent, while (2)H NMR data were highly sorbose dependent.     

Determination of Ploidy Level and Nuclear DNA Content in Tunisian Populations of Atriplex halimus L.

Atriplex halimus L. (Chenopodiaceae) (Saltbush) is a perennial species used as a fodder shrub for livestock in arid and semi-arid areas, particularly in North Africa. The aim of this work was to determine whether differences in ploidy level and/or nuclear DNA content exist among populations from widely-separated sites in Tunisia. We determined nuclear DNA content and chromosome numbers for populations of A. halimus from seven different locations (Gabes, Medenine, Tataouine, Monastir, Tunis, Sidi Bouzid, Kairouan). The chromosome counts showed that all the Tunisian populations, plus a population from Eraclea (Italy), were tetraploid (2n = 4x = 36) whereas a population from Cala Tarida (Spain) was diploid (2n = 2x = 18). With respect to nuclear DNA, the 2C DNA content of population Cala Tarida was estimated to be 2.41 pg. There was no significant difference among the tetraploid populations (or among plants within populations), whose 2C DNA content ranged from 4.92 to 4.97 pg.