Rocío Reina

Role of arbuscular mycorrhizal fungus Rhizophagus custos in the dissipation of PAHs under root-organ culture conditions

Polycyclic aromatic hydrocarbons (PAHs) are one of the most common contaminants in soil. Arbuscular mycorrhizal (AM) fungi make host plants resistant to pollutants. This study aims to evaluate the impact of anthracene, phenanthrene and dibenzothiophene on the AM fungus Rhizophagus custos, isolated from soil contaminated by heavy metals and PAHs, under monoxenic conditions. We found a high level of tolerance in R. custos to the presence of PAHs, especially in the case of anthracene, in which no negative effect on AM-colonized root dry weight (root yield) was observed, and also a decrease in the formation of anthraquinone was detected. Increased PAH dissipation in the mycorrhizal root culture medium was observed; however, dissipation was affected by the level of concentration and the specific PAH, which lead us to a better understanding of the possible contribution of AM fungi, and in particular R. custos, to pollutant removal.

Solid state fermentation of olive mill residues by wood- and dung-dwelling Agaricomycetes: Effects on peroxidase production, biomass development and phenol phytotoxicity

The in vivo conversion of dry olive mill residue (DOR) by wood- and dung-dwelling fungi - Auricularia auricula-judae, Bjerkandera adusta and Coprinellus radians - increases peroxidase secretion up to 3.2-3.5-fold (∼1.3, 3.5 and 7.0 Ug(-1) DOR for dye-decolorizing peroxidase, manganese peroxidase and aromatic peroxygenases, respectively). The incubation of DOR with these fungi produced a sharp decrease in total phenolic content (100% within 4 wk), a reduction in phytotoxicity as well as a certain degree of plant growth caused by the stimulating effect of fungal-treated DOR. These findings correlate with a characteristic shift in the fragmentation pattern of water-soluble aromatics (detected at 280 nm) from low (0.2, 1.5 and 2.2 kDa, respectively) to high molecular mass (35 to >200 kDa), which demonstrates the presence of a polymerization process. Phenol-rich agricultural residues are a useful tool for enzyme expression and production studies of peroxidase-producing Agaricomycetes which could make DOR a valuable organic fertilizer.

Differences in the secretion pattern of oxidoreductases from Bjerkandera adusta induced by a phenolic olive mill extract

The secretome of the white-rot fungus Bjerkandera adusta produced in synthetic Kirk medium was compared to that supplemented with an aqueous phenol-rich extract of dry olive mill residues (ADOR). Distinct changes in the protein composition of oxidoreductases, namely diverse class-II peroxidases and aryl alcohol oxidases were found. In the ADOR-supplemented medium (ASC), 157 distinct proteins were identified by the secretome analysis, whereas only 59 of them were identified without ADOR supplementation (Kirk medium culture; KM). Proteome analysis indicated that the number of peroxidases produced in ASC was more than doubled (from 4 to 11) compared to KM. Two short manganese peroxidases (MnP1 and MnP6) and one versatile peroxidase (VP1) represented 29% of the relative abundance (NSAF) in ASC. Two of them (MnP1 and VP1) were also detected in KM at a relative abundance (NSAF) of only 3%. Further peroxidases present in ASC were one lignin peroxidase (LiP2), one generic peroxidase (GP) and three dye-decolorizing peroxidases (DyPs). The relative abundance of DyPs and aryl alcohol oxidases (AAO) were lower in ASC in comparison to KM. In addition to peptide sequence analysis, the secretion of Mn(2+)-oxidizing peroxidases as well as AAOs were followed by enzyme measurement. The Mn(2+)-oxidizing activity increased nearly 30-fold (from 10 to 281Ul(-1)) after ADOR addition. Two enzymes responsible for that activity were successfully purified (BadVPI and BadVPII). To prove a potential involvement of these enzymes in the degradation of aromatic compounds, BadVPI was tested for its ability to degrade the recalcitrant dehydrogenated polymer (DHP, synthetic lignin). These results show that natural phenol-rich materials act as secretome-stimulating additives. Applying these substances enables us to investigate fungal degradation and detoxification processes and gives more insight into the complexity of fungal secretomes, e.g. of white-rot fungi.

Reduced dry olive residue phytotoxicity in the field by the combination of physical and biological treatments

Olive oil extraction generates large amounts of olive mill residues (DOR) which may be used as organic fertilizer. The influence of a combination of physical fractionation and saprobe fungal incubation on the phytotoxicity of DOR was studied. The physical fractions of DOR, obtained following extraction using ethyl acetate (EDOR) and wa- ter (ADOR) were less phytotoxic than DOR with respect to the shoot dry weight of to- mato. There was no relationship between the total phenol content of the different DOR physical fractions and their phytotoxicity. The saprophytic fungus Coriolopsis rigida reduced DOR and ADOR phytotoxicity and eliminated the phytotoxicity of EDOR. However, unlike the physical treatments of DOR, the decrease in the phenol content of EDOR and ADOR caused by C. rigida was closely paralleled to the decrease in their phytotoxicity. After 30 days of incubation, C. rigida was able to eliminate the phytotoxicity of EDOR on tomato plants grown in the field.

An Overview of Fungal Applications in the Valorization of Lignocellulosic Agricultural By-Products: The Case of Two-Phase Olive Mill Wastes

DOR (dry mill olive residue) is an agricultural by-product generated during two-phase olive oil extraction. It is a microtoxic and phytotoxic phenolic-rich lignocellulosic residue and is produced in high amounts in Mediterranean countries. Several techniques have been proposed for its valorization. Agaricomycetes are one of the most efficient lignin-modifying enzyme (LME) producers on Earth (e.g. laccases, peroxidases or the novel peroxygenases). The secretion of these enzymes varies according to the specific strain and also depends on other factors such as the presence of certain elicitors in the medium, like lignocellulosic materials or phenols, which may be crucial to stimulate LME secretion. Due to the enzymatic capability of fungi, numerous studies have been performed in order to valorize the DOR, such as its recent use as a medium for enzyme production or organic soil amendment. An improved valorization of these by-products and a better understanding of fungal enzyme production could lead to the development of biorefineries that utilize various components in biomass and their intermediates, thus maximizing the value derived from biomass feedstock. In this chapter, we summarize some studies performed on this topic (mainly based on the usage of white-rot fungi in the transformation of DOR), and we discuss possible trends, challenges and future prospects for the use of fungi in an environmentally sustainable scenario.

Interactions between phenolic compounds present in dry olive residues and the arbuscular mycorrhizal symbiosis

The use of “alpeorujo” (dry olive residue) has been proposed as an organic amendment in order to enhance soil structure and to increase C storage in soils. The aim of this work is to study how aqueous alpeorujo (ADOR) extracts bioremediated with white-rot fungi and three representative phenolic acids present in this extract (protocatechuic, vanillic and caffeic acid) affect the growth of the arbuscular mychorrhizal fungus Rhizophagus custos in monoxenic culture. Our results show that ADOR decreased mycorrhization parameters; however, this negative effect ceased after ADOR bioremediation. Although protocatechuic and vanillic acids have drastic negative effects at high concentrations, these phenols enhance mycorrhization processes at low concentrations and caffeic acid negatively affects symbiosis at low concentrations. Finally, the capacity of root biomass to dissipate individual phenols was also estimated, in which mycorrhized roots improve phenol dissipation in the growth medium in the presence of different phenols. This study highlights the important role played by arbuscular mycorrhiza in protecting plants from phytotoxicity.

Effect of DOR Incubated with Saprobe Fungi on Hydrolytic Enzymes Activities and Chemical Properties of Rhizospheric Soil of Lettuce

The dry olive residue (DOR), a by-product of the olive mill industry, is produce in large quantities in Mediterranean countries. The phytotoxic and antimicrobial properties of this residue adversely impact on soil qualities. The objective of this study was to investigate the evolution of soil enzymes activities (acid phosphatase, β-glucosidase, protease and urease) and chemical properties (pH, phenols, total organic carbon (TOC) and soluble carbohydrates) after the agronomic application on lettuce of either un-treated DOR or DOR incubated with inmobilized Panus tigrinus or Fusarium lateritium. A decrease of total phenols and phytotoxicity on lettuce plants in presence of treated DOR related to the un-treated residue was detected. We observed a decrease of all hydrolytic enzymes, except protease, after the soil incubation with un-treated DOR for 15 days. It appears that the high concentration of phenolics compounds inhibited the activities of these enzymes. However, the microbial activity was stimulated by the addition of DOR incubated with the saprobe fungi as indicated by the increase of soil enzyme activities detected. The protease activity was always higher than the control soil irrespective of the type of amendant. The TOC and phenols of rhizospheric soil of lettuce increased after 15 and 30 days of agronomic application of untreated or treated residue and a general decrease of the soluble carbohydrates contents was found after 30 days of soil incubation. The DOR detoxified by saprobe fungi has been seen to have a positive effect to restore the loss rhizospheric soil functionality detected after the addition of un-treated residue.