Alessandro D’Annibale

Reduction of the phenolic components in olive-mill wastewater by an enzymatic treatment and its impact on durum wheat (Triticum durum Desf.) germinability

Olive-mill wastewater (OMW), an effluent of olive oil extraction process, is annually produced in huge amounts in olive growing areas. An interesting option for its disposal is the spreading on agricultural land, provided that phytotoxic effects are neutralized. The objective of the present investigation was to evaluate the potential of an enzyme-based treatment in removing OMW phytotoxicity. To this aim, germinability experiments on durum wheat (Triticum durum Desf. cv. Duilio) were conducted in the presence of different dilutions of raw or enzyme-treated OMW. OMW treatment with laccase resulted in a 65% and 86% reduction in total phenols and ortho-diphenols respectively, due their polymerization as revealed by size-exclusion chromatography. Raw OMW exerted a significant concentration-dependent inhibition on the germinability of durum wheat seeds which was evident up to a dilution rate of 1:8. When the effluent was treated with a fungal laccase, germinability was increased by 57% at a 1:8 dilution and by 94% at a 1:2 dilution, as compared to the same dilutions using untreated OMW. The treatment with laccase also decreased the mean germination time by about 1 day as compared to untreated controls. These results show that germinability inhibition due to OMW can be reduced effectively using fungal laccase, suggesting that phenols are the main determinants of its phytotoxicity.

Oxirane-immobilized Lentinula edodes laccase: stability and phenolics removal efficiency in olive mill wastewater.

Immobilization of Lentinula edodes laccase on Eupergit C increased pH, thermal and proteolytic stability with slight modifications in laccase oxidation efficiency. Immobilized laccase proved to be efficiently stable in removing olive mill wastewater phenolics.

Characterization of immobilized laccase from Lentinula edodes and its use in olive-mill wastewater treatment

This study is mainly concerned with the characterization of an immobilized laccase from the white-rot fungus Lentinula edodes for use in wastewater treatment. The enzyme was immobilized on chitosan by adsorption and subsequent cross-linking with glutaraldehyde. The immobilized laccase displayed a lower specific activity and a lower substrate affinity than the free enzyme. Nevertheless, the immobilized catalyst exhibited an appreciable catalytic capability (520 units g−1 support) along with remarkably improved stability properties to various parameters, such as temperature, pH and storage time. The treatment of olive mill wastewater with immobilized laccase led to a partial decolorization as well as to significant abatements in its content in polyphenols, ortho-diphenols combined with a decreased toxicity of the effluent.

The biodegradation of recalcitrant effluents from an olive mill by a white-rot fungus

Biodegradation of olive-mill wastewater (OMW) was performed by the polyurethane-immobilized mycelium of Lentinula edodes. Throughout three consecutive treatment cycles of the effluent significant abatements of its polluting characteristics were observed. In fact, its contents in total organic carbon, total phenols, total ortho-diphenol were dramatically reduced. In addition, a significant effluent decolorization was evident.

Assessment of olive-mill wastewater as a growth medium for lipase production by Candida cylindracea in bench-top reactor.

Olive-mill wastewater (OMW) was investigated for its suitability to serve as a medium for lipase production by Candida cylindracea NRRL Y-17506. The OMW that best supported enzyme production was characterized by low COD and low total sugars content. In shake flask batch cultures, OMW supplementation with 2.4 g l(-1) NH(4)Cl and 3 g l(-1) olive oil led to an enzyme activity of about 10 U ml(-1). The addition of glucose or malt extract and supplements containing organic N (e.g., peptone, yeast extract) either depressed or did not affect the enzyme production. Further experiments were then performed in a 3-l stirred tank reactor to assess the impact of medium pH and stirring speed on the yeast enzyme activity. The lipase activity was low (1.8 U ml(-1)) when the pH was held constant at 6.5, significantly increased (18.7 U ml(-1)) with uncontrolled pH and was maximum (20.4 U ml(-1)) when the pH was let free to vary below 6.5. A stirring regime, that varied depending on the dissolved oxygen concentration in the medium, both prevented the occurrence of anoxic conditions during the exponential growth phase and enabled good lipase production (i.e., 21.6 U ml(-1)) and mean volumetric productivity (i.e., 123.5 Ul(-1)h(-1)).

Addition of allochthonous fungi to a historically contaminated soil affects both remediation efficiency and bacterial diversity.

Botryosphaeria rhodina DABAC P82 and Pleurotus pulmonarius CBS 664.97 were tested for their ability to grow and to degrade aromatic hydrocarbons in an aged contaminated soil. To evaluate the impact of indigenous microflora on the overall process, incubations were performed on both fumigated and nonfumigated soils. Fungal colonization by B. rhodina was unexpectedly lower in the fumigated than in the nonfumigated soil while the growth of P. pulmonarius showed an opposite response. Degradation performances and detoxification by both fungi in the nonfumigated soil were markedly higher than those observed in the fumigated one. Heterotrophic bacterial counts in nonfumigated soil augmented with either B. rhodina or P. pulmonarius were significantly higher than those of the corresponding incubation control (6.7 ± 0.3 × 108 and 8.35 ± 0.6 × 108, respectively, vs 9.2 ± 0.3 × 107). Bacterial communities of both incubation controls and fungal-augmented soil were compared by numerical analysis of denaturing gradient gel electrophoresis profiles of polymerase chain reaction (PCR)-amplified 16S ribosomal RNA (rRNA) genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Besides increasing overall diversity, fungal augmentation led to considerable qualitative differences with respect to the pristine soil.

Bioremediation of long-term PCB-contaminated soil by white-rot fungi

The objective of this work was to test the PCB-degrading abilities of two white-rot fungi, namely Pleurotus ostreatus and Irpex lacteus, in real contaminated soils with different chemical properties and autochthonous microflora. In addition to the efficiency in PCB removal, attention was given to other important parameters, such as changes in the toxicity and formation of PCB transformation products. Moreover, structural shifts and dynamics of both bacterial and fungal communities were monitored using next-generation sequencing and phospholipid fatty acid analysis. The best results were obtained with P. ostreatus, which resulted in PCB removals of 18.5, 41.3 and 50.5% from the bulk, top (surface) and rhizosphere, respectively, of dumpsite soils after 12 weeks of treatment. Numerous transformation products were detected (hydoxylated and methoxylated PCBs, chlorobenzoates and chlorobenzyl alcohols), which indicates that both fungi were able to oxidize and decompose the aromatic moiety of PCBs in the soils. Microbial community analysis revealed that P. ostreatus efficiently colonized the soil samples and suppressed other fungal genera. However, the same fungus substantially stimulated bacterial taxa that encompass putative PCB degraders. The results of this study finally demonstrated the feasibility of using this fungus for possible scaled-up bioremediation applications.

Effect of Mobilising Agents on Mycoremediation of Soils Contaminated by Hydrophobic Persistent Pollutants

The last decades have witnessed an increasing concern of the public opinion and competent authorities regarding the accumulation of hydrophobic persistent pollutants (HPP) in both natural and anthropic ecosystems. An emerging restoration approach to contaminated soils, often referred to as bioremediation, involves the use of either resident or exogenous microbes. Its efficacy, however, is strongly affected by the recalcitrance of HPP to microbial attack due to their low water solubility and bioavailability. In this respect, the use of surface-active agents able to enhance the mobilisation of these contaminants from the soil organic phase to the aqueous one has been often shown to stimulate their biodegradation. Although the HPP degradation capability of filamentous fungi has been suggested to be less dependent on bioavailability than other microbiota, there is increasing evidence of the use of mobilising agents in fungal-assisted remediation applications. This review analyses the influence of both surfactants and other pollutant-mobilising agents, such as plant oils and cyclodextrins, on mycoremediation performances with reference to their impact on fungal growth, physiology and interactions with resident soil communities.

Isolation and characterization of lignin from beech wood and chestnut sawdust for the preparation of lignin nanoparticles (LNPs) from wood industry side-streams

Abstract Lignin was isolated through mild acidolysis from local wood sources such as beech wood and chestnut wood sawdust, a high-volume side product of wood industries. The lignin fractions were characterized by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), Fourier-transform infrared (FTIR), ultraviolet-visible (UV-Vis) and two-dimensional heteronuclear single-quantum correlation nuclear magnetic resonance (2D HSQC NMR) spectroscopies and size exclusion chromatography (SEC). The Klason lignin (KL) content and polysaccharide composition were determined using traditional methods. Lignin nanoparticles (LNPs) were prepared via a non-solvent method involving dialysis and characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and FTIR and UV-VIS spectroscopies. Semi-porous as well as hollow nanoparticles endowed with a spherical shape were observed. The large majority of the LNPs exhibited an average particle diameter of 90–120 nm. Dynamic light scattering (DLS) analysis showed that both distribution and frequency of dimensional classes of LNPs are clearly affected by the lignin solvent system, i.e. solvent selection governs the size distribution of LNPs.

Pleurotus ostreatus biofilms exhibit higher tolerance to toxicants than free-floating counterparts

The MBECTM-High Throughput Assay based on the Calgary Biofilm Device was used to produce and to characterize Pleurotus ostreatus biofilms. Hydroxyapatite coating of pegs was required to enable biofilm attachment; biofilm amounts and homogeneity of distribution were markedly improved upon removal of non-sessile biomass after 48 h from inoculation. Scanning electron microscopy showed surface-associated and multi-layered growth stabilized by the presence of an extracellular matrix (ECM). Biofilms had higher contents of total sugars and ECM than their free-floating counterparts. Tolerance to Cr(VI) in the former was about twice that of the latter as inferred by the respective inhibitory concentrations (48.4 vs 24.1 mM and 114.5 vs 61.0 mM in 4- and 7-d-old cultures, respectively). Biofilms also displayed superior olive-mill wastewater (OMW) treatment efficiency along 5 consecutive batches leading to chemical oxygen demand and total phenol removals higher than 50 and 90%, respectively. Laccase activity peaks in biofilm cultures grown on OMW were significantly higher than those in free-floating cultures.