Main Factors Determining the Scale-Up Effectiveness of Mycoremediation for the Decontamination of Aliphatic Hydrocarbons in Soil.

Soil contamination constitutes a significant threat to the health of soil ecosystems in terms of complexity, toxicity, and recalcitrance. Among all contaminants, aliphatic petroleum hydrocarbons (APH) are of particular concern due to their abundance and persistence in the environment and the need of remediation technologies to ensure their removal in an environmentally, socially, and economically sustainable way. Soil remediation technologies presently available on the market to tackle soil contamination by petroleum hydrocarbons (PH) include landfilling, physical treatments (e.g., thermal desorption), chemical treatments (e.g., oxidation), and conventional bioremediation. The first two solutions are costly and energy-intensive approaches. Conversely, bioremediation of on-site excavated soil arranged in biopiles is a more sustainable procedure. Biopiles are engineered heaps able to stimulate microbial activity and enhance biodegradation, thus ensuring the removal of organic pollutants. This soil remediation technology is currently the most environmentally friendly solution available on the market, as it is less energy-intensive and has no detrimental impact on biological soil functions. However, its major limitation is its low removal efficiency, especially for long-chain hydrocarbons (LCH), compared to thermal desorption. Nevertheless, the use of fungi for remediation of environmental contaminants retains the benefits of bioremediation treatments, including low economic, social, and environmental costs, while attaining removal efficiencies similar to thermal desorption. Mycoremediation is a widely studied technology at lab scale, but there are few experiences at pilot scale. Several factors may reduce the overall efficiency of on-site mycoremediation biopiles (mycopiles), and the efficiency detected in the bench scale. These factors include the bioavailability of hydrocarbons, the selection of fungal species and bulking agents and their application rate, the interaction between the inoculated fungi and the indigenous microbiota, soil properties and nutrients, and other environmental factors (e.g., humidity, oxygen, and temperature). The identification of these factors at an early stage of biotreatability experiments would allow the application of this on-site technology to be refined and fine-tuned. This review brings together all mycoremediation work applied to aliphatic petroleum hydrocarbons (APH) and identifies the key factors in making mycoremediation effective. It also includes technological advances that reduce the effect of these factors, such as the structure of mycopiles, the application of surfactants, and the control of environmental factors.

Mixed glycerol and orange peel-based substrate for fed-batch microbial biodiesel production.

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 (r L) of 0.20 g L-1 h-1 and YL/X of 0.80; the DSF-FB process yielded LVP, r L 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.

Development of laboratory-scale sequential electrokinetic and biological treatment of chronically hydrocarbon-impacted soils.

This study focused on the remediation of a chronically diesel-polluted soil by combining an electrokinetic treatment with a variety of bioremediation approaches. Priority within the sequential treatment was given to electrokinetic remediation (EKR) since the application of natural attenuation (NA), biostimulation and site-specific bio-augmentation resulted in very low degradation performance for total petroleum hydrocarbons (TPH) and polycyclic hydrocarbons (PAH). The application of 20-day EKR (1.0 V cm-1 with polarity reversal) led to 47.2 % and 46.2 % removal of TPH and PAH, respectively, and exerted a negative impact on bacterial abundance, as determined indirectly by quantitative PCR of 16S rRNA genes and community function, as investigated by community-level physiological profiling. These adverse effects were transient and, after a 50-day NA treatment applied downstream from EKR, bacterial abundance was an order of magnitude higher than that found in the initial soil and TPH and PAH removals were significantly higher than those attained by EKR (64.1 % and 56.3 %, respectively). The combination of EKR with site-specific bioaugmentation led to the greatest TPH and PAH degradation (76.0 % and 78.6 %, respectively). The results indicate that bioremediation can be successfully applied downstream from EKR and that the adverse effects exerted by this physico-chemical approach on soil microbiota are reversible.

Orange peel waste-based liquid medium for biodiesel production by oleaginous yeasts.

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.

Mechanisms of arsenic assimilation by plants and countermeasures to attenuate its accumulation in crops other than rice.

Arsenic is a ubiquitous metalloid in the biosphere, and its origin can be either geogenic or anthropic. Four oxidation states (-3, 0, +3 and + 5) characterize organic and inorganic As- compounds. Although arsenic is reportedly a toxicant, its harmful effects are closely related to its chemical form: inorganic compounds are most toxic, followed by organic ones and finally by arsine gas. Although drinking water is the primary source of arsenic exposure to humans, the metalloid enters the food chain through its uptake by crops, the extent of which is tightly dependent on its phytoavailability. Arsenate is taken up by roots via phosphate carriers, while arsenite is taken up by a subclass of aquaporins (NIP), some of which involved in silicon (Si) transport. NIP and Si transporters are also involved in the uptake of methylated forms of As. Once taken up, its distribution is regulated by the same type of transporters albeit with mobility efficiencies depending on As forms and its accumulation generally occurs in the following decreasing order: roots > stems > leaves > fruits (seeds). Besides providing a survey on the uptake and transport mechanisms in higher plants, this review reports on measures able to reducing plant uptake and the ensuing transfer into edible parts. On the one hand, these measures include a variety of plant-based approaches including breeding, genetic engineering of transport systems, graft/rootstock combinations, and mycorrhization. On the other hand, they include agronomic practices with a particular focus on the use of inorganic and organic amendments, treatment of irrigation water, and fertilization.

Time-Dependent Changes in Morphostructural Properties and Relative Abundances of Contributors in Pleurotus ostreatus/Pseudomonas alcaliphila Mixed Biofilms.

Pleurotus ostreatus dual 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 of P. 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 using Pseudomonas alcaliphila 34 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.

Aspergillus olivimuriae sp. nov., a halotolerant species isolated from olive brine.

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.

Preparation of Lignin Nanoparticles from Wood Waste for Wood Surface Treatment.

Lignin was isolated from wood wastes comprising Iroko sawdust (IR) and mixed sawdust from Iroko and Norway spruce (IRNS), furnished by a local wood houses producer. The respective acidolysis lignin fractions were structurally characterized using pyrolysis (Py)-GCMS, two-dimensional heteronuclear single quantum correlation nuclear magnetic resonance (2D HSQC NMR), Fourier-transform infrared FTIR and ultraviolet-visible (UV-VIS) spectroscopies, size exclusion chromatography, and standard wet-chemistry methods for Klason lignin and polysaccharides determination. The isolated lignin fractions were subsequently used for the preparation of lignin nanoparticles (LNPs) using a non-solvent method. LNPs were then used for wood surface treatment using a dip-coating technique. The coated wood samples were analyzed by colorimetry and scanning electron microscopy (SEM) before and after artificial weathering experiments in a UV chamber to investigate the UV protection potential of the LNPs coatings. Wood samples dip-coated with LNPs showed promising surface modifications resembling a sort of film of fused LNPs. Coatings made from IR-LNPs and IRNS-LNPs performed significantly better in artificial weathering experiments than uncoated reference samples.

Production of lignin-modifying enzymes by Trametes ochracea on high-molecular weight fraction of olive mill wastewater, a byproduct of olive oil biorefinery.

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.

Impact of the Fenton-like treatment on the microbial community of a diesel-contaminated soil.

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.

Fungal Community Structure and As-Resistant Fungi in a Decommissioned Gold Mine Site.

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 were Mucor and Mortierella. The relative abundances of Basidiomycota, instead, tended to raise as the contamination increased; within this phylum the most abundant genera were Cryptococcus and Pseudotomentella. 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, and Chaetomium), 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.

A sustainable use of Ricotta Cheese Whey for microbial biodiesel production.

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.

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.

Comparative assessment of fungal augmentation treatments of a fine-textured and historically oil-contaminated soil.

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 60days, 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 CO2 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.

Assessment of degradation potential of aliphatic hydrocarbons by autochthonous filamentous fungi from a historically polluted clay soil.

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.

Implications of polluted soil biostimulation and bioaugmentation with spent mushroom substrate (Agaricus bisporus) on the microbial community and polycyclic aromatic hydrocarbons biodegradation.

Different applications of spent Agaricus bisporus substrate (SAS), a widespread agro-industrial waste, were investigated with respect to the remediation of a historically polluted soil with Polycyclic Aromatic Hydrocarbons (PAH). In one treatment, the waste was sterilized (SSAS) prior to its application in order to assess its ability to biostimulate, as an organic amendment, the resident soil microbiota and ensuing contaminant degradation. For the other treatments, two bioaugmentation approaches were investigated; the first involved the use of the waste itself and thus implied the application of A. bisporus and the inherent microbiota of the waste. In the second treatment, SAS was sterilized and inoculated again with the fungus to assess its ability to act as a fungal carrier. All these treatments were compared with natural attenuation in terms of their impact on soil heterotrophic and PAH-degrading bacteria, fungal growth, biodiversity of soil microbiota and ability to affect PAH bioavailability and ensuing degradation and detoxification. Results clearly showed that historically PAH contaminated soil was not amenable to natural attenuation. Conversely, the addition of sterilized spent A. bisporus substrate to the soil stimulated resident soil bacteria with ensuing high removals of 3-ring PAH. Both augmentation treatments were more effective in removing highly condensed PAH, some of which known to possess a significant carcinogenic activity. Regardless of the mode of application, the present results strongly support the adequacy of SAS for environmental remediation purposes and open the way to an attractive recycling option of this waste.

Screening, isolation, and characterization of glycosyl-hydrolase-producing fungi from desert halophyte plants.

Fungal strains naturally occurring on the wood and leaves of the salt-excreting desert tree Tamarix were isolated and characterized for their ability to produce cellulose- and starch-degrading enzymes. Of the 100 isolates, six fungal species were identified by ITS1 sequence analysis. No significant differences were observed among taxa isolated from wood samples of different Tamarix species, while highly salt-tolerant forms related to the genus Scopulariopsis (an anamorphic ascomycete) occurred only on the phylloplane of T. aphylla. All strains had cellulase and amylase activities, but the production of these enzymes was highest in strain D, a Schizophyllum-commune-related form. This strain, when grown on pretreated Tamarix biomass, produced an enzymatic complex containing levels of filter paperase (414 +/- 16 IU/ml) that were higher than those of other S. commune strains. The enzyme complex was used to hydrolyze different lignocellulosic substrates, resulting in a saccharification rate ofpretreated milk thistle (73.5 +/- 1.2%) that was only 10% lower than that obtained with commercial cellulases. Our results support the use of Tamarix biomass as a useful source of cellulolytic and amylolytic fungi and as a good feedstock for the economical production of commercially relevant cellulases and amylases.

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

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.

Characterization of Pleurotus ostreatus biofilms by using the calgary biofilm device.

The adequacy of the Calgary biofilm device, often referred to as the MBEC system, as a high-throughput approach to the production and subsequent characterization of Pleurotus ostreatus biofilms was assessed. The hydroxyapatite-coating of pegs was necessary to enable biofilm attachment, and the standardization of vegetative inocula ensured a uniform distribution of P. ostreatus biofilms, which is necessary for high-throughput evaluations of several antimicrobials and exposure conditions. Scanning electron microscopy showed surface-associated growth, the occurrence of a complex aggregated growth organized in multilayers or hyphal bundles, and the encasement of hyphae within an extracellular matrix (ECM), the extent of which increased with time. Chemical analyses showed that biofilms differed from free-floating cultures for their higher contents of total sugars (TS) and ECM, with the latter being mainly composed of TS and, to a lesser extent, protein. Confocal laser scanning microscopy analysis of 4-day-old biofilms showed the presence of interspersed interstitial voids and water channels in the mycelial network, the density and compactness of which increased after a 7-day incubation, with the novel occurrence of ECM aggregates with an α-glucan moiety. In 4- and 7-day-old biofilms, tolerance to cadmium was increased by factors of 3.2 and 11.1, respectively, compared to coeval free-floating counterparts.

Addition of maize stalks and soybean oil to a historically PCB-contaminated soil: effect on degradation performance and indigenous microbiota.

Objective of this study was to assess the single or combined effect of a plant oil and a lignocellulosic waste, namely soybean oil (SO) and maize stalks (MS), respectively, on resident microbiota and bioremediation performances of a soil historically contaminated by medium to highly chlorinated PCBs. Higher concentrations of both biphenyl- and chlorobenzoate-degrading cultivable bacteria were found in the MS-amended microcosms (MSM) than the non amended or SO-amended ones after 30 d incubation at 28°C. Fungal growth, instead, was strikingly stimulated in the microcosms that had undergone concomitant MS and SO supplementation (MS-SOM). Denaturing gradient gel electrophoresis analyses of 16S and 18S rRNA genes showed that both amendments promoted a remarkable increase in both bacterial and fungal biodiversity. The abundances of biphenyl-2,3-dioxygenase (bph) and that of catechol-2,3-dioxygenase (C230) genes in the non-amended contaminated soil were constant over time. Conversely, after 60 d incubation, bph and C230 abundances increased 2.8- and 61-fold in the MSM, respectively, and, in the MS-SOM, 1.4- and 46-fold, respectively, with respect to the zero time point. Although the overall PCB removal was not positively affected by the amendments, the concomitant presence of both MS and SO led to significantly higher depletions of hexa-, hepta-, octa- and nona-chlorinated congeners than in the non-amended microcosms (i.e. 24.6, 22, 20.5 and 9.5%, versus 19.4, 16.4, 14.7 and 6.1%, respectively). In all microcosms, PCB degradation was negatively correlated with hydrophobicity, organic matter/water partition coefficient, molecular weight and extent of chlorination of the pollutants with the notable exception of the MS-SOM ones where such a relationship was less stringent.