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LIFE MySOIL project presentation delivered by Fiora Bagnato, from Eni Rewind, and Davide Lelli, from the University of La Tuscia (UNITUS) during Ecomondo 2022, which was held between November 7th-10th, 2022, in Rimini (Italy)., This project has received funding from the LIFE Programme of the European Commission under contract number LIFE20 ENV/ES/000416.

Orange peel waste (OPW), the primary byproduct of the juice extraction process, is annually generated in massive amounts (21 Mton), and its aqueous extraction in biorefining operations yields a liquid fraction, referred to as orange peel extract (OPE). Although OPE contains significant amounts of easily assimilable carbohydrates, such as fructose, glucose, and sucrose, no investigations have been conducted yet to assess its possible use in biodiesel production by oleaginous yeasts. Consequently, the objective of the present study was to assess whether OPE might act as the basis of a liquid medium for microbial lipid production. A screening conducted with 18 strains of oleaginous yeasts in shaken flask on the OPE-based medium showed that Rhodosporidium toruloides NRRL 1091 and Cryptococcus laurentii UCD 68-201 gave the best results in terms of lipid production (5.8 and 4.5 g L−1, respectively) and accumulation (77 and 47% on a dry matter basis, respectively). The subsequent scale transfer of the process to a 3-L STR operated in batch mode halved the time required to reach the lipid peak with the ensuing increase in volumetric productivities in R. toruloides NRRL 1091 (3646 mg L−1 day−1) and C. laurentii UCD 68-201 (2970.7 mg L−1 day−1). The biodiesel yields from the lipids of the former and the latter strain were 36.9 and 31.9%, respectively. Based on multivariate analysis of fatty acid methyl ester compositions, the lipids from the former and the latter strain were highly resembling those of Jatropha and palm oils, two commonly used feedstocks for biodiesel manufacturing.

A facultative halo-tolerant Aspergillus strain was isolated from olive brine waste, the effluent from the debittering process of table olives. Phenotypic and molecular characteristics showed clearly that the isolate represents a novel species. Based on the source of isolation, the new species has been named Aspergillus olivimuriae. It was found tolerant to high concentrations of NaCl (15 %) or sucrose (60 %) and it exhibits substantial growth under these conditions. Although the new species grew profusely at 37 °C, no growth was observed at 40 °C, conidia en masse were avellaneous on all media. The description of the new species Aspergillus olivimuriae brings the total species of Aspergillus sect. Flavipedes to 15. The type strain of A. olivimuriae sp. nov. is NRRL 66783 (CCF 6208), its whole genome has been deposited as PRJNA498048.

The aqueous extraction of orange peel waste (OPW), the byproduct of the juice extraction process generated annually in massive amounts (21 Mton), yields a carbohydrate-rich liquid fraction, termed orange peel extract (OPE). Several studies highlight that the combination of glycerol, a biodiesel byproduct, with carbohydrate mixtures might boost microbial lipid production. This study performed first a shaken flask screening of 15 oleaginous yeast strains based on their growth and lipid-producing abilities on OPE- and glycerol-based media. This screening enabled the selection of R. toruloides NRRL 1091 for the assessment of the process transfer in a stirred tank reactor (STR). This assessment relied, in particular, on either single- and double-stage feeding fed-batch (SSF-FB and DSF-FB, respectively) processes where OPE served as the primary medium and nitrogen-containing glycerol-OPE mixtures as the feeding one. The continuous supply mode at low dilution rates (0.02 and 0.01 h−1 for SSF-FB and DSF-FB, respectively) starting from the end of the exponential growth of the initial batch phase enabled the temporal extension of biomass and lipid production. The SSF-FB and DSF-FB processes attained high biomass and lipid volumetric productions (LVP) and ensured significant lipid accumulation on a dry cell basis (YL/X). The SSF-FB process led to LVP of 20.6 g L−1 after 104 h with volumetric productivity (rL) of 0.20 g L−1 h−1 and YL/X of 0.80; the DSF-FB process yielded LVP, rL and YL/X values equal to 15.92 g L−1, 0.11 g L−1 h−1 and 0.65, respectively. The fatty acid profiles of lipids from both fed-batch processes were not significantly different and resembled that of Jatropha oil, a vastly used feedstock for biodiesel production. These results suggest that OPE constitutes an excellent basis for the fed-batch production of R. toruloides lipids, and this process might afford a further option in OPW-based biorefinery., Biotechnology; Microbiology; Waste Treatment; Green Engineering; Sustainable Development; Microbial Biotechnology; Biofuel; orange peel waste; Biodiesel; fed-batch process; Rhodosporidium toruloides; oleaginous yeasts; Circular economy.

Microbial oils are regarded as a sustainable alternative to vegetable oils for biodiesel manufacturing. However, in order to develop a cost-effective process, high-lipid producer microorganisms should be combined with low-cost renewable growth substrates. For this reason, the objective of the present study was to assess comparatively the oil-producing performance of 9 oleaginous fungi belonging to the Aspergillus, Mucor, Mortierella and Cunninghamella genera on three relevant and widespread waste, such as glycerol, orange peel extract (OPE) and ricotta cheese whey (RCW). This screening was performed at the shaken flask level and, among the strains under study, Mortierella isabellina NRRL 1757 turned out to be the most efficient and versatile and its lipid profile was found to be highly compatible with biodiesel production. Process transfer of M. isabellina lipid production to the lab-scale Stirred Tank Reactor on all the three waste-based media, was shown to be feasible, achieving a lipid productivity of 0.46, 1.24 and 0.91 g/(L d) on glycerol, OPE and RCW, respectively. Noteworthy, the fatty acid analysis of the oils produced, confirmed their suitability for biodiesel manufacturing, exhibiting a high similarity to palm and Jatropha oils commonly used as feedstock for this production.

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.

This is the final version of the following paper: CROGNALE S, STAZI SR, FIRRINCIELI A, PESCIAROLI L, FEDI S, PETRUCCIOLI M, D'ANNIBALE A 2019. Time-dependent changes in morpho-structural properties and relative abundances of contributors in Pleurotus ostreatus/Pseudomonas alcalliphila mixed biofilms. Frontiers in Microbiology, 10, p.1819 (ISSN: 1664-302X) DOI: https://doi.org/10.3389/fmicb.2019.01819 Scopus ID: 2-s2.0-85072733107 Web of Science ID: 000480249900001 PubMed ID: 31447819 URL of published article: https://www.frontiersin.org/articles/10.3389/fmicb.2019.01819/full, This study was supported by the Ministero dell'Istruzione dell'Università e della Ricerca (MIUR) within the project 2008P7K379. This work was also partially supported by the "Departments of Excellence-2018" Program (Dipartimenti di Eccellenza) of the Italian Ministry of Education, University and Research (MIUR), DIBAF-Department of University of Tuscia, Project "Landscape 4.0 – food, wellbeing, and environment".

Pleurotus ostreatusdual biofilms with bacteria are known to be involved in rock phosphate solubilization, endophytic colonization, and even in nitrogen fixation. Despite these relevant implications, no information is currently available on the architecture ofP. ostreatus-based dual biofilms. In addition to this, there is a limited amount of information regarding the estimation of the temporal changes in the relative abundances of the partners in such binary systems. To address these issues, a dual biofilm model system with this fungus was prepared by usingPseudomonas alcaliphila34 as the bacterial partner due to its very fast biofilm-forming ability. The application of the bacterial inoculum to already settled fungal biofilm on a polystyrene surface coated with hydroxyapatite was the most efficient approach to the production of the mixed system the ultrastructure of which was investigated by a multi-microscopy approach. Transmission electron microscopy analysis showed that the adhesion of bacterial cells onto the mycelial cell wall appeared to be mediated by the presence of an abundant layer of extracellular matrix (ECM). Scanning electron microscopy analysis showed that ECM filaments of bacterial origin formed initially a reticular structure that assumed a tabular semblance after 72 h, thus overshadowing the underlying mycelial network. Across the thickness of the mixed biofilms, the presence of an extensive network of channels with large aggregates of viable bacteria located on the edges of their lumina was found by confocal laser scanning microscopy; on the outermost biofilm layer, a significant fraction of dead bacterial cells was evident. Albeit with tangible differences, similar results regarding the estimation of the temporal shifts in the relative abundances of the two partners were obtained by two independent methods, the former relying on qPCR targeting of 16S and 18S rRNA genes and the latter on ester-linked fatty acid methyl esters analysis.

This is the accepted manuscript of the paper "Identification of Resistance Genes and Response to Arsenic in Rhodococcus aetherivorans BCP1", published as final paper in "Frontiers in Microbiology Volume 10, 07 May 2019, Pages 888 https://doi.org/10.3389/fmicb.2019.00888”. Arsenic (As) ranks among the priority metal(loid)s that are of public health concern. In the environment, arsenic is present in different forms, organic or inorganic, featured by various toxicity levels. Bacteria have developed different strategies to deal with this toxicity involving different resistance genetic determinants. Bacterial strains of Rhodococcus genus, and more in general Actinobacteria phylum, have the ability to cope with high concentrations of toxic metalloids, although little is known on the molecular and genetic bases of these metabolic features. Here we show that Rhodococcus aetherivorans BCP1, an extremophilic actinobacterial strain able to tolerate high concentrations of organic solvents and toxic metalloids, can grow in the presence of high concentrations of As(V) (up to 240 mM) under aerobic growth conditions using glucose as sole carbon and energy source. Notably, BCP1 cells improved their growth performance as well as their capacity of reducing As(V) into As(III) when the concentration of As(V) is within 30–100 mM As(V). Genomic analysis of BCP1 compared to other actinobacterial strains revealed the presence of three gene clusters responsible for organic and inorganic arsenic resistance. In particular, two adjacent and divergently oriented ars gene clusters include three arsenate reductase genes (arsC1/2/3) involved in resistance mechanisms against As(V). A sequence similarity network (SSN) and phylogenetic analysis of these arsenate reductase genes indicated that two of them (ArsC2/3) are functionally related to thioredoxin (Trx)/thioredoxin reductase (TrxR)-dependent class and one of them (ArsC1) to the mycothiol (MSH)/mycoredoxin (Mrx)-dependent class. A targeted transcriptomic analysis performed by RT-qPCR indicated that the arsenate reductase genes as well as other genes included in the ars gene cluster (possible regulator gene, arsR, and arsenite extrusion genes, arsA, acr3, and arsD) are transcriptionally induced when BCP1 cells were exposed to As(V) supplied at two different sub-lethal concentrations. This work provides for the first time insights into the arsenic resistance mechanisms of a Rhodococcus strain, revealing some of the unique metabolic requirements for the environmental persistence of this bacterial genus and its possible use in bioremediation procedures of toxic metal contaminated sites. &nbsp

The production of seed oils from Cynara cardunculus generates huge amounts of lignocellulosic residues which can be exploited according to a cascade approach. In this paper, residual cardoon biomass (RCB) was tested as a growth substrate for the solid-state production of cellulolytic cocktails by species known to produce glucose-tolerant β-glucosidase isoenzymes. Best productions were obtained with 10-d-old Aspergillus tubingensis cultures on RCB supplemented with wheat bran (200 g kg−1) yielding β-glucosidase and endo-β-1,4-glucanase activities as high as (25 and 4) IU g−1, respectively, and 4 FPU g−1. The saccharification performance of the obtained cocktail tested on acid-catalysed steam-exploded RCB at low solid loading (25 g dm−3) was around 53% at 20 FPU g−1 cellulose. These performance were significantly enhanced by adding the xylanase-rich NS 22083 commercial formulation, reaching glucose yields higher than 80% after 72 h incubation. The use of the catalytic additive was optimized by a statistical approach, based on factorial analysis. A comparison of the performance of the A. tubingensis reinforced cocktail with the Cellic®CTec2 taken as benchmark formulation was done at the same enzyme load and performed at industrially relevant solid loadings, namely at (100 and 200) g dm−3. This comparison showed that Cellic®CTec2 led to only slightly higher glucose yields while an opposite outcome was observed for xylose yields, irrespective of the solid loading conditions. Thus, this study shows that an in-house enzyme production, based on the solid-state conversion of an industrial byproduct, able of yielding cellulolytic cocktails with substantial saccharification performance is feasible.

The removal of aged hydrophobic contaminants from fine-textured soils is a challenging issue in remediation. The objective of this study was to compare the efficacy of augmentation treatments to that of biostimulation in terms of total aliphatic hydrocarbon (TAH) and toxicity removal from a historically contaminated clay soil and to assess their impact on the resident microbial community. To this aim, Pleurotus ostreatus , Botryosphaeria rhodina and a combination of both were used as the inoculants while the addition of a sterilized lignocellulose mixture to soil (1:5, w / w ) was used as a biostimulation approach. As opposed to the non-amended control soil, where no changes in TAH concentration and residual toxicity were observed after 60 days, the activation of specialized bacteria was found in the biostimulated microcosms resulting in significant TAH removal (79.8%). The bacterial community structure in B. rhodina -augmented microcosms did not differ from the biostimulated microcosms due to the inability of the fungus to be retained within the resident microbiota. Best TAH removals were observed in microcosms inoculated with P. ostreatus alone ( Po ) and in binary consortium with B. rhodina (BC) (86.8 and 88.2%, respectively). In these microcosms, contaminant degradation exceeded their bioavailability thresholds determined by sequential supercritical CO 2 extraction. Illumina metabarcoding of 16S rRNA gene showed that the augmentation with Po and BC led to lower relative abundances of Gram(+) taxa, Actinobacteria in particular, than those in biostimulated microcosms. Best detoxification, with respect to the non-amended incubation control, was found in Po microcosms where a drop in collembola mortality (from 90 to 22%) occurred. At the end of incubation, in both Po and BC, the relative abundances of P. ostreatus sequences were higher than 60% thus showing the suitability of this fungus in bioaugmentation-based remediation applications.

Arsenic (As) contamination in drinking water represents a worldwide threat to human health. During last decades, the exploitation of microbial As-transformations has been proposed for bioremediation applications. Among biological methods for As-contaminated water treatment, microbial As(III)-oxidation is one of the most promising approaches since it can be coupled to commonly used adsorption removal technologies, without requiring the addition of chemicals and producing toxic by-products. Despite the As(III) oxidation capability has been described in several bacterial pure or enrichment cultures, very little is known about the real potentialities of this process when mixed microbial communities, naturally occurring in As contaminated waters, are used. This study highlighted the contribution of native groundwater bacteria to As(III)-oxidation in biofilters, under conditions suitable for a household-scale treatment system. This work elucidated the influence of a variety of experimental conditions (i.e., various filling materials, flow rates, As(III) inflow concentration, As(III):As(V) ratio, filter volumes) on the microbially-mediated As(III)-oxidation process in terms of oxidation efficiency and rate. The highest oxidation efficiencies (up to 90% in 3 h) were found on coarse sand biofilters treating total initial As concentration of 100 μg L−1. The detailed microbial characterization of the As(III) oxidizing biofilms revealed the occurrence of several OTUs affiliated with families known to oxidize As(III) (e.g., Burkholderiaceae, Comamonadaceae, Rhodobacteraceae, Xanthomonadaceae). Furthermore, As-related functional genes increased in biofilter systems in line with the observed oxidative performances.

The high-molecular weight fraction of olive mill wastewater (HMW-OMW), a byproduct of olive oil biorefinery, was used at the reactor level as the basal medium for production of laccase and Mn-dependent peroxidase (MnP) by Trametes ochracea. Three reactor systems, namely stirred tank reactors equipped with either Rushton turbines or marine impeller and draft tube (STR and STR-MD, respectively) and an air-lift reactor (ALR) were compared for this purpose. Although inocula were supplied as intact pellets, in both STR-based systems fungal growth evolved rapidly into a dispersed form while the ALR enabled the maintenance of the pellet growth mode. STR was deemed to be the most promising system since it best supported the production MnP activity on the HMW-OMW-based medium and its performance in laccase production did not differ from that observed with the STR-MD. Among the stirring regimes considered (250, 400, 500 and 600 rpm), the best production in the STR was observed at 500 rpm and 1.0 vvm for both laccase (8850 ± 270 IU L−1 on day 15) and MnP (17,027.4 ± 87.2 IU L-1 on day 13). When the inocula were supplied to the STR in homogenized form, the MnP production peak (16,856 ± 1070 IU L-1) was attained 8 days earlier than the previous condition and that of laccase was nearly doubled (14,967 ± 907 IU L-1). When compared with literature data, T. ochracea MnP production and productivity on the HMW-OMW-based medium were the highest reported for a wild-type fungal strain.

Arundo donax L. has received much attention from researchers as biomass crop because of its vigorous growth, high productivity and low agronomic inputs requirements. Once harvested, the chopped biomass of A. donax L. may be stored for a variable period according to energy demand or logistic reasons. As consequence of storage, the chopped biomass may undergo processes of degradation that can compromise the energy yield of the product and the economic convenience of the cultivation. In the view of preserving the A. donax L. biomass for producing different kind of energy, such as bio-ethanol, biogas, and fuel for combustion, the aim of this study was to investigate, in small-scale, different storage systems assessing the dynamics of storage and their effects on energy losses and fuel quality. The work has included different studies, such as (i) the monitoring of heat development, moisture content and dry matter losses, (ii) the analysis of polymeric compounds and combustion performances. Results indicate that according to the storage method adopted, the conservation of the product can be significantly influenced, with clear impacts on its energy potential. Polymeric composition changed after storage according to the method used, while energetic characteristics were not remarkably affected.

A strain of a fungal species, i.e., Dichostereum sordulentum, not yet isolated from either Uruguay or decayed Eucalyptus spp. wood, was investigated for its ability of producing extracellular ligninolytic enzymes. To this aim, the strain was grown on a N-rich liquid medium added with either bark or wood Eucalyptus dunnii sawdust. The former additive was more effective than the latter instimulating laccase leading to a production of 4.78 IU mL-1 which was further enhanced by the concomitant presence of 80 ppm CuSO4 (6.56 IU mL-1). The two order of magnitude higher proanthocyanidin content in bark than in wood suggests that this fraction might be responsible for the higher stimulatory effect of the former on laccase production.

Naphthenic acids (NAs) are an important group of toxic organic compounds naturally occurring in hydrocarbon deposits. This work shows that Rhodococcus aetherivorans BCP1 cells not only utilize a mixture of eight different NAs (8XNAs) for growth but they are also capable of marked degradation of two model NAs, cyclohexanecarboxylic acid (CHCA) and cyclopentanecarboxylic acid (CPCA) when supplied at concentrations from 50 to 500 mgL-1. The growth curves of BCP1 on 8XNAs, CHCA, and CPCA showed an initial lag phase not present in growth on glucose, which presumably was related to the toxic effects of NAs on the cell membrane permeability. BCP1 cell adaptation responses that allowed survival on NAs included changes in cell morphology, production of intracellular bodies and changes in fatty acid composition. Transmission electron microscopy (TEM) analysis of BCP1 cells grown on CHCA or CPCA showed a slight reduction in the cell size, the production of EPS-like material and intracellular electron-transparent and electron-dense inclusion bodies. The electron-transparent inclusions increased in the amount and size in NA-grown BCP1 cells under nitrogen limiting conditions and contained storage lipids as suggested by cell staining with the lipophilic Nile Blue A dye. Lipidomic analyses revealed significant changes with increases of methyl-branched (MBFA) and polyunsaturated fatty acids (PUFA) examining the fatty acid composition of NAs-growing BCP1 cells. PUFA biosynthesis is not usual in bacteria and, together with MBFA, can influence structural and functional processes with resulting effects on cell vitality. Finally, through the use of RT (Reverse Transcription)-qPCR, a gene cluster (chcpca) was found to be transcriptionally induced during the growth on CHCA and CPCA. Based on the expression and bioinformatics results, the predicted products of the chcpca gene cluster are proposed to be involved in aerobic NA degradation in R. aetherivorans BCP1. This study provides first insights into the genetic and metabolic mechanisms allowing a Rhodococcus strain to aerobically degrade NAs.

Although large quantities of heavy metal laden wastes are released in an uncontrolled manner by gold mining activities with ensuing contamination of the surrounding areas, there is scant information on the mycobiota of gold-mine sites. Thus, the present study was aimed to describe the fungal community structure in three differently As- and Hg-polluted soils collected from the Pestarena decommissioned site by using Illumina®metabarcoding. Fungal richness was found to increase as the contamination level increased while biodiversity was not related to the concentrations of inorganic toxicants. Within the phylum Zygomigota which, irrespective of the contamination level, was predominant in all the soils under study, the most abundant genera wereMucorandMortierella. The relative abundances of Basidiomycota, instead, tended to raise as the contamination increased; within this phylum the most abundant genera wereCryptococcusandPseudotomentella. The abundance of Ascomycota, ranging from about 8 to 21%, was not related to the contamination level. The relative abundances of those genera (i.e.,Penicillium, Trichoderma, andChaetomium), the cultivable isolates of which exhibited significant As-resistance, were lower than the set threshold (0.5%). Mass balances obtained from As-exposure experiments with these isolates showed that the main mechanisms involved in counteracting the toxicant were accumulation and, above all, volatilization, the respective extents of which ranged from 0.6 to 5.9% and from 6.4 to 31.2% in dependence of the isolate.

Fenton-like treatment (FLT) is an ISCO technique relying on the iron-induced H2O2 activation in the presence of additives aimed at increasing the oxidant lifetime and maximizing iron solubility under natural soil pH conditions. The efficacy of FLT in the clean-up of hydrocarbon-contaminated soils is well established at the field-scale. However, a better assessment of the impact of the FLT on density, diversity and activity of the indigenous soil microbiota, might provide further insights into an optimal combination between FLT and in-situ bioremediation (ISB). The aim of this work was to assess the impacts of FLT on the microbial community of a diesel-contaminated soil collected nearby a gasoline station. Different FLT conditions were tested by varying either the H2O2 concentrations (2 and 6%) or the oxidant application mode (single or double dosage). The impact of these treatments on the indigenous microbial community was assessed immediately after the Fenton-like treatment and after 30, 60 and 90 d and compared with enhanced natural attenuation (ENA). After FLT, a dramatic decrease in bacterial density, diversity and functionality was evident. Although in microcosms with double dosing at 2% H2O2 a delayed recovery of the indigenous microbiota was observed as compared to those subjected to single oxidant dose, after 60 d incubation the respiration rate increased from 0.036 to 0.256 μg CCO2 g-1soil h-1. Irrespective of the oxidant dose, best degradation results after 90 d incubation (around 80%) were observed with combined FLT, relying on double oxidant addition, and bioremediation.

Groundwater plays a central role in the hydrological cycle and represents the utmost natural resource for human consumption and activities on a global scale. Therefore, any source of contamination of either geogenic or anthropogenic origin may provide a serious environmental health threat. Within the long list of organic and inorganic groundwater contaminants, arsenic, a toxic element retrieved in air, soils, rocks, waters and organisms, can occur at high concentrations in aquifers representing an issue of worldwide concern. Over the past years, research efforts aimed to elucidate the microorganisms and mechanisms involved in the biogeochemical cycling of this element. An emerging challenge is to identify and exploit microbial metabolic potentialities for arsenic-contaminated water treatment. The objective of this review is to outline the existing knowledge about ecology, biochemistry and genomics of arsenic-related microorganisms, with particular reference to their distribution and their capabilities to oxidize As(III) in groundwater. Moreover, a broad evaluation of the application potentialities of microbiological processes suitable for treatment strategies of arsenic-contaminated groundwater is provided.

The development of rapid techniques, such as hyperspectral spectrophotometry, for investigating arsenic (As) soil contamination could be of great value with respect to conventional methods. This study was conducted to detect As concentrations in artificially polluted soils (from 25 to 1045 mg kg−1) through hyperspectral visible–near infrared spectrophotometry and to compare two multivariate statistical regression analyses: partial least squares and support vector machines. The correlation coefficient r is greater in the partial least squares in both model (0.93%) and test (0.87%) with respect to support vector machines (0.88% for the model and 0.82% for the test). The most important model variables extracted from the variable importance in projection scores resulted the absorption peaks at 580, 660, 715, and 780 nm. Bands in the visible spectra are not directly associated to As, but the metalloid can interact with the main spectrally active components of soil permitting to multivariate statistical models ...

An experimental biofuel wood chip storage site was studied, as a potential fungal “reservoir,” by means of quantitative and qualitative assessments of airborne fungal spores. Fungal load in the bio-aerosol, determined through active and passive methods, declined with the distance from wood piles. Occupational exposure was comparatively evaluated when two specific operational tasks, manual and mechanized handling, were performed. Under the conditions tested, the manual operators were more exposed to the airborne fungal spores (4864 ± 580 CFU m−3 air). The collected spores were identified as belonging to species of the genera Alternaria, Aspergillus, Penicillium, Fusarium, Cladosporium, Pleospora, Cochliobolus, Epicoccum, Absidia, and Trichoderma. Most prevalent were the genera Alternaria and Cladosporium, with the highest percentages of occurrence (30 and 12%, respectively). To the best of our knowledge, this is the first work reporting the identification through molecular methods of airborne fungi released during the handling of wood chip biofuel biomass.

Orange peel waste (OPW) was converted into ethanol by consecutive acid-catalysed steam-explosion (ACSE), enzymatic saccharification and fermentation with Saccharomyces cerevisiae F15. The first step was performed in the presence of 0.5% (volume fraction) sulphuric acid at a solid loading of 160 g L−1 using a novel laboratory-scale Direct Steam-Injection Apparatus under different reaction temperatures and times. ACSE at 200 °C for 90 s (or 180 °C for 150 s) yielded the highest solubilization of pectin (∼73%) and a positive effect on the subsequent enzymatic hydrolysis step, that was carried out at 50 °C with an enzyme loading of 6 FPU g−1 cellulose. OPW, pretreated at 180 °C for 150 s, yielded the highest glucose solubilisation degree (∼56%) at the end of the saccharification step, and the maximum ethanol yield coefficient (∼0.495 g g−1) and productivity (4.85 g L−1 h−1) at the end of the 3rd repeated fermentation batch in shaken-flasks.

Three different Tamarix species, namely Tamarix aphylla, T. aphylla “Erect”-type and Tamarix jordanis, were grown in an experimental field under extreme desert conditions and irrigated with either reclaimed sewage or brackish water. Depending on both species and source of irrigation, the above-ground biomass production ranged from 18 to 36 Mg ha−1 in the first year. Among the three chemically characterized Tamarix species, T. jordanis was selected due to its higher cellulose content, and lower hemicellulose and phenol contents so as to outline a preliminary process flow sheet for ethanol production. This included steam-injection heating under acidic conditions (200 °C; 90 s; 0.5% H2SO4; 160 g L−1 solid loading) using a novel lab-scale Direct Steam Injection Apparatus, enzymatic saccharification (50 °C; pH 5.0; 200 g L−1 solid loading; 20 FPU g−1 cellulose) and subsequent ethanolic fermentation (30 °C; Saccharomyces cerevisiae F-15 as the inoculum). Lab-scale fermentation runs were carried out in a 3-L stirred bioreactor in repeated-batch mode and showed an almost quantitative conversion of glucose into ethanol (0.507 ± 0.006 g g−1), thus leading to a satisfactory overall process ethanol yield of about 145 L Mg−1 Tamarix biomass.

The MBEC(TM)-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.

An industrial three-cascade-reactor plant treating 45 m3 d−1 of dairy wastewater (DW) was monitored for approx. one year to investigate the effect of variable daily influent loads. It removed more than 85% COD, NH4-N and non-ionic and anionic surfactants from DW within the loads 7–24, 0.4–2.3, 0.4–0.7 and 0.1–0.5 kg d−1, respectively; NH4-N removal, in particular, was almost quantitative. Although the degradation of the above parameters below the lower load thresholds declined to 78.7, 87.5, 50.2 and 64.7%, respectively, their residual concentrations met effluent discharge standards. The biomass settling properties, assessed as sludge volume index (SVI), were satisfactory (generally lower than 150 ml g−1) regardless of the organic load of the influent. The depletion of the pollutant load took mainly place in the first reactor albeit a significant contribution to the removal of the slowly degradable organic matter fraction was given by the two subsequent reactors.

The increasing demand of plant oils for biodiesel production has highlighted the need for alternative strategies based either on non-food crops or agro-industrial wastes that do not compete with food and feed production. In this context, the combined use of wastewater and oleaginous microorganisms could be a valuable production option. Ricotta cheese whey (RCW), one of the major byproducts of the dairy industry, is produced in very high and steadily increasing amounts and, due to its high organic load, its disposal is cost-prohibitive. In the present study, in order to assess the adequacy of RCW as a growth medium for lipid production, 18 strains of oleaginous yeasts were investigated in shaken flask for their growth and lipid-producing capabilities on this substrate. Among them, Cryptococcus curvatus NRRL Y-1511 and Cryptococcus laurentii UCD 68-201 adequately grew therein producing substantial amounts of lipids (6.8 and 5.1gL-1, respectively). A high similarity between the percent fatty acid methyl esters (FAME) composition of lipids from the former and the latter strain was found with a predominance of oleic acid (52.8 vs. 48.7%) and of total saturated fatty acids (37.9 vs. 40.8%). The subsequent scale transfer of the C. laurentii UCD 68-201 lipid production process on RCW to a 3-L STR led to significantly improved biomass and total lipid productions (14.4 and 9.9gL-1, respectively) with the biodiesel yield amounting to 32.6%. Although the C. laurentii FAME profile was modified upon process transfer, it resembled that of the Jatropha oil, a well established feedstock for biodiesel production. In conclusion, C. laurentii UCD 68-201, for which there is very limited amount of available information, turned out to be a very promising candidate for biodiesel production and wide margins of process improvement might be envisaged.

The main aim of this study was to assess the ability of producing extracellular phenoloxidases (EPO) of fungi isolated from olive brine wastewater (OBW), the effluent from the debittering process of table olives. Five out of twenty isolates displayed EPO-producing ability as assessed by selective agar plate assays and were all halotolerant. Among them, Citeromyces matritensis (syn. Candida globosa) and Aspergillus fumigatus concomitantly produced laccase and Mn-peroxidase (MnP) activities. Candida boidinii and Candida bombi, instead, only produced laccase and Candida diddensiae only released MnP. Both C. matritensis and A. fumigatus, grown in shaken cultures on OBW, maintained their EPO-producing ability and removed phenols by 52.3 and 82.3%, respectively, after 10 d incubation in the non-supplemented effluent. These results might suggest the possible use of these strains in the treatment of other saline phenol-rich effluents, such as pickling and tannery wastewater.

The low moisture content of the dry olive mill residue (DOR), solid waste from the two-phase olive oil extraction process, enables its long-term storage and its ready reconstitution with water thus leading to a candidate liquid growth medium for the microbial production of enzymes of commercial interest. To test this hypothesis, two white-rot fungi (WRF), namely Lentinus tigrinus CBS 577.79 and Phlebia sp. DABAC 9, were grown in shaken cultures on non-supplemented aqueous extracts of the dry olive mill residue (ADOR) and compared for their ability to grow therein and produce Mn-dependent peroxidase (MnP). Then, in order to assess the feasibility of scale transfer to the reactor level, the two fungi were grown in a 3-L bubble-column reactor on 25% (w/v) ADOR at 28 °C and an aeration rate of 0.3 vvm. Under these conditions, L. tigrinus and Phlebia sp. produced 42.60 ± 1.74 and 38.88 ± 1.68 nkatal mL −1 MnP with respective mean volumetric productivities of 222.9 and 324.1 nkatal L −1 h −1 . These results clearly show that the use of non-supplemented ADOR as a production medium enables a significant MnP production thus representing an excellent basis for process optimization.

In this work, a novel laboratory-scale direct steam-injection apparatus (DSIA) was developed to overcome the main drawback of the conventional batch-driven lab rigs, namely the long time needed to heat fiber slurry from room to reaction temperatures greater than 150 °C. The novel apparatus mainly consisted of three units: (i) a mechanically-stirred bioreactor where saturated steam at 5-30 bar can be injected; (ii) an automatic on-off valve to flash suddenly the reaction medium after a prefixed reaction time; (iii) a cyclone separator to recover the reacted slurry. This system was tested using 0.75 dm³ of an aqueous solution of H₂SO₄ (0.5%, v/v) enriched with 50 kg m⁻³ of either commercial particles of Avicel® and Larch xylan or 0.5 mm sieved particles of Tamarix jordanis. Each slurry was heated to about 200 °C by injecting steam at 28 bar for 90 s. The process efficiency was assessed by comparing the dissolution degree of suspended solid (Y(S)), as well as xylose (Y(X)), glucose (Y(G)), and furfural (Y(F)) yields, with those obtained in a conventional steam autoclave at 130 °C for 30 or 60 min. Treatment of T. jordanis particles in DSIA resulted in Y(S) and Y(G) values quite similar to those obtained in the steam autoclave at 130 °C for 60 min, but in a less efficient hemicellulose solubilization. A limited occurrence of pentose degradation products was observed in both equipments, suggesting that hydrolysis predominated over degradation reactions. The susceptibility of the residual solid fractions from DSIA treatment to a conventional 120 h long cellulolytic treatment using an enzyme loading of 5.4 FPU g⁻¹ was markedly higher than that of samples hydrolysed in the steam autoclave, their corresponding glucose yields being equal to 0.94 and 0.22 g per gram of initial cellulose, respectively. Thus, T. jordanis resulted to be a valuable source of sugars for bioethanol production as proved by preliminary tests in the novel lab rig developed here.

The short-term response of the resident soil bacterial and fungal communities to the addition of 5% (w/w) of either dry olive mill residue (DOR), DOR treated with Phlebia sp. (PTDOR) or DOR previously extracted with water (WEDOR) was investigated. As opposed to bacteria, the diversity of fungi increased upon the amendments as assessed by denaturing gradient gel electrophoresis of 18S rDNA. Over the first 30 days, phospholipid fatty acids analyses indicated a gradual decrease in the relative abundances of Gram+ bacteria (from 44.8% to 37.9%) and a concomitant increase of Gram- bacteria (from 37.3% to 51.2%) in DOR-amended soil. A considerable increase in the fungal/bacterial ratio was observed after 7 days in DOR, WEDOR and PTDOR-amended soils with respect to the control (0.316, 0.165 and 0.265, respectively, vs. 0.011). The overall microbial activity was stimulated by the amendments as indicated by the higher activity levels of both dehydrogenase and fluorescein diacetate hydrolase. These results indicate that DOR at the application level examined is not toxic on soil microorganisms. L'articolo é disponibile sul sito dell'editore: http://www.sciencedirect.com

BACKGROUND: Mobilizing agents (MAs) have been suggested to improve the fungal degradation of polycyclic aromatic hydrocarbons (PAHs) in soil. Three different MAs (Tween 20, Tween 80 and soybean oil) were investigated for their ability to stimulate contaminant degradation by either Phlebia sp. DABAC 9 or Allescheriella sp. DABAC1 in a soil spiked with a mixture of PAHs. RESULTS: Phlebia sp. and Allescheriella sp. markedly differed in their growth capabilities under non-sterile conditions and without MAs (3.0 versus 0.1 µg ergosterol g−1 soil, respectively). However, soybean oil led to a 35-fold increase of Allescheriella sp. growth. Contaminant degradations by Phlebia sp. DABAC 9 and Allescheriella sp. DABAC 1 were best supported by soybean oil and Tween 20, respectively. Enumeration of cultivable bacteria and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA showed that microbial density and biodiversity were positively affected by the mycoremediation especially with Allescheriella sp., the use of which led to an evident detoxification. CONCLUSIONS: Allescheriella sp. DABAC 1 appears to be a promising strain in the remediation of PAH-contaminated soils. The different response of the two fungi to MAs addition confirms the stringent need for a preliminary lab-scale assessment of fungus/MA combinations prior to application. Copyright © 2009 Society of Chemical Industry

Dry olive mill residue (DOR), the by-product of the two-phase extraction process, is very rich in organic matter and nutritionally relevant cations. For this reason, the agronomic use of this waste has been suggested although DOR exhibits significant phytotoxicity. The objective of this study was to investigate the impact of Paecilomyces farinosus on both organic matter modification and detoxification of this waste.Humification ratio in DOR colonized by the fungus for 20 weeks was increased by about 65% with respect to the abiotic control and humification index reached 0.38, a value that characterizes well-humified materials. High performance size-exclusion chromatography of humic acids from fungal cultures showed a marked increase in both weight-averaged and number-averaged molecular weights with respect to abiotic controls. Water-soluble phenols were reduced by 45% in 20-week-old P. farinosus cultures on DOR and mass-balance ultra-filtration showed that the relative abundance of the molecular weight fraction of phenols above 30 kDa increased from 31 to 72% suggesting the occurrence of polymerization.Experiments performed with alfalfa grown on soils containing 2.5% (w/w) of abiotic controls and fungaltreated DOR showed that phytotoxicity was totally suppressed in the waste that underwent fungal treatment. L'articolo è disponibile sul sito dell'editore: http://www.sciencedirect.com

The impact of several mobilizing agents (MAs) (i.e., soybean oil, Tween-20, Tween-80, olive-oil mill wastewaters, and randomly methylated beta-cyclodextrins) on the degradation performances of the white-rot fungi Irpex lacteus and Pleurotus ostreatus was comparatively assessed in a soil spiked with a mixture of seven polycyclic aromatic hydrocarbons (PAHs). Among the different MAs, soybean oil best supported the growth of both fungi that was twice that observed in soil in the absence of MAs. In addition, soybean oil positively affected PAH degradation by both fungi. In this case, the total weight of organic contaminants (TWOC) was lower than that in the absence of MAs (57.7 vs. 201.3 and 26.3 vs. 160.4 mg kg(-1) with I. lacteus and P. ostreatus, respectively). On the other hand, the number of cultivable heterotrophic bacteria was significantly lower in the soil with soybean oil augmented with either one of the two fungi (5.21 vs. 8.71 and 0.22 vs. 0.51 x 10(7) CFU g(-1) soil with I. lacteus and P. ostreatus, respectively). The effect of soybean oil was confirmed by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes that showed a general decrease in biodiversity. The impact of the other MAs on bacterial diversity was either slightly negative or positive in incubation controls. Both richness and Shannon-Weaver index decreased upon treatment with P. ostreatus. Moreover, with this fungus the composition of the indigenous bacteria was not significantly affected by the type of MA used. By contrast, both indices increased in soil with I. lacteus in the presence of randomly methylated beta-cyclodextrins (39 vs. 33 and 1.43 vs. 1.26, respectively) and soybean oil (19 vs. 5 and 1.01 vs. 0.65, respectively).

Penicillium variabile P16 is characterized by the ability to release high levels of glucose oxidase when grown in glucose-rich media converting, at the same time, glucose into gluconic acid. This work reports on gluconic acid production by the above strain grown in bench-top reactor under different strategies, traditional batch and fed-batch processes; Na2CO3 was substituted for CaCO3 as the buffering agent in order to prevent calcium gluconate precipitation over the mycelium that would limit oxygen uptake and, as a consequence, glucose conversion. Best results were obtained with an improved fed-batch process which, in addition to glucose, was continuously fed with nitrogen and minerals at a flow rate of 0.02 ml min−1: yield and total productivity were 99.4% and 2.02 g h−1, respectively, and remained constant till the end of the fermentation (240 h).

In this work the production of an extra-cellular β-glucan by Botryosphaeria rhodina DABAC P82, previously studied in a conventional bench-top stirred bioreactor, was enhanced by resorting to two different strategies consisting of dispersing different volumetric fractions of paraffin oil into the culture broth or replacing the six flat-blade Rushton turbines with 45° pitched four-blade open style (fan) turbines with different impeller-to-vessel diameter ratios (D/T). Use of fan turbines with D/T = 0.54 at a tip impeller speed of 1.4 m s−1 resulted in less viscous culture broths and most advantageous values of the β-glucan concentration (18.7 ± 0.3 kg m−3), and yield coefficients for β-glucan on mycelial biomass formed (5.1 ± 0.8 g g−1) and specific power consumed (23.3 ± 0.4 g W−1).

Fumaric acid production by Rhizopus arrhizus from commercial hydrolysates of corn starch (i.e. glucose molasses) was studied at different initial concentrations of glucose (S) and C:N ratios (R) by performing a 3(2) factorial experiment. By using the response surface methodology and statistical analysis, fumaric acid (YF) and mycelial biomass (YX) yields, as referred to the initial concentration of glucose and fumaric acid productivity (PF), were fitted to the only significant first-order effects of S and R with mean percentage errors ranging from 11 to 15%. The resulting empiric models were used to determine the optimal values of S (100-130 g dm-3) and R (150-210 g-atom C per g-atom N) associated with YF and PF varying in the ranges 40-49% and 7-8.5 g dm-3 day-1, respectively. After establishing the validity of these data at the 95% confidence level, an optimal operating condition (S = 120 g dm-3 and R = 150) was further tested using other substrates (i.e. glucose and acid or enzymatic hydrolysates of cassava, corn and potato flours). Statistically significant improvements in the fumaric acid yield and productivity were determined with respect to the predicted values. Since the highest values of YF and PF were obtained from the acid hydrolysates of the starch-based materials and such values were also found to be insensitive to the substrate used (at a probability level of 0.05), the above operating condition might be further employed to minimise fumaric acid production costs as a function of the feedstock used.

Bioavailability of pollutants in contaminated soils represents an important factor influencing the bioremediation process. We investigated the impact of the bioavailability of polycyclic aromatic hydrocarbons (PAHs) on the efficacy of mycoremediation of aged matrices. Two historically contaminated soils, differing in both overall PAH concentrations (2320 vs. 612 mg PAHs kg - 1 ) and in the relative amount of the non-bioavailable fraction (4.0 vs. 32%, respectively), were incubated with two white rot fungi – Irpex lacteus and Pleurotus ostreatus. When relating biodegradation results with the amount of PAH bioavailable fractions estimated by sequential supercritical fluid extractions, the correlation coefficients ranged from 0.60 to 0.83. In order to assess a possible increase of the mycoremediation efficiency, four different mobilizing agents (MAs), i.e. soybean oil, Tween 20, Tween 80 and olive-mill wastewater, were added to soil prior to fungal incubations. The addition of MAs to the soil with a negligible amount of the non-bioavailable fraction had either a limited or even a negative impact on PAH degradation. The same was true also in the case of the other soil, characterized by a larger non-bioavailable fraction. Both white rot fungi under study markedly degraded PAHs in historically contaminated soils differing significantly in PAH content and the bioavailability. However, both the fungi performed better in the soil with higher pollutant bioavailability. Consequently, a preliminary determination of non-bioavailable fraction might provide a useful indication of the performance of bioremediation treatment.

Thermophilic bacteria have been isolated from several terrestrial, marine and industrial environments. Anaerobic digesters treating organic wastes are often an important source of these microorganisms, which catalyze a wide array of metabolic processes. Moreover, organic wastes are primarily composed of proteins, whose degradation is often incomplete. Coprothermobacter spp. are proteolytic anaerobic thermophilic microbes identified in several studies focused on the analysis of the microbial community structure in anaerobic thermophilic reactors. They are currently classified in the phylum Firmicutes; nevertheless, several authors showed that the Coprothermobacter group is most closely related to the phyla Dictyoglomi and Thermotoga. Since only a few proteolytic anaerobic thermophiles have been characterized so far, this microorganism has attracted the attention of researchers for its potential applications with high-temperature environments. In addition to proteolysis, Coprothermobacter spp. showed several metabolic abilities and may have a biotechnological application either as source of thermostable enzymes or as inoculum in anaerobic processes. Moreover, they can improve protein degradation by establishing a syntrophy with hydrogenotrophic archaea. To gain a better understanding of the phylogenesis, metabolic capabilities and adaptations of these microorganisms, it is of importance to better define the role in thermophilic environments and to disclose properties not yet investigated.

The present work was aimed at isolating and identifying the main members of the mycobiota of a clay soil historically contaminated by mid- and long-chain aliphatic hydrocarbons (AH) and to subsequently assess their hydrocarbon-degrading ability. All the isolates were Ascomycetes and, among them, the most interesting was Pseudoallescheria sp. 18A, which displayed both the ability to use AH as the sole carbon source and to profusely colonize a wheat straw:poplar wood chip (70:30, w/w) lignocellulosic mixture (LM) selected as the amendment for subsequent soil remediation microcosms. After a 60 d mycoaugmentation with Pseudoallescheria sp. of the aforementioned soil, mixed with the sterile LM (5:1 mass ratio), a 79.7% AH reduction and a significant detoxification, inferred by a drop in mortality of Folsomia candida from 90 to 24%, were observed. However, similar degradation and detoxification outcomes were found in the non-inoculated incubation control soil that had been amended with the sterile LM. This was due to the biostimulation exerted by the amendment on the resident microbiota, fungi in particular, the activity and density of which were low, instead, in the non-amended incubation control soil.

Glucose oxidase (GOX) is a glycoprotein that finds wide application in food industry and clinical analysis. The gene encoding the GOX from Penicillium variabile P16 was expressed in Pichia pastoris X 33 using the methanol inducible AOX1 promoter. Among 11 transformants resistant toward high zeocin concentrations, six Mut+ strains were screened in shaken flasks and the strain X33 c9, producing 0.33 U ml−1 of heterologous GOX after 11 days of fermentation, was selected. Recombinant GOX (ca. 50 U ml−1) was produced in a 3-l fermenter under not optimized conditions, recovered and purified in order to characterize and to compare it with the native one. The GOX from P. pastoris had a molecular weight of 82 kDa. Comparison of carbohydrate moieties showed a slight over-glycosylation of the GOX from Pichia over the native enzyme (17 and 14%, respectively). pH behavior of the recombinant enzyme, in terms of both activity and stability, was similar to that of the native one; on the other hand, a certain difference was observed in optimal temperature for activity and in thermal stability. P. pastoris appears to be a good expression system for GOX production.