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The present study investigated the valorization of olive-mill wastewater (OMW) by its use as a possible growth medium for the microbial production of extra-cellular lipase. To this end, strains of Geotrichum candidum (NRRL Y-552 and Y-553), Rhizopus arrhizus (NRRL 2286 and ISRIM 383), Rhizopus oryzae (NRRL 6431), Aspergillus oryzae (NRRL 1988 and 495), Aspergillus niger (NRRL 334), Candida cylindracea (NRRL Y-17506) and Penicillium citrinum (NRRL 1841 and 3754, ISRIM 118) were screened. All strains were able to grow on the undiluted OMW, producing extra-cellular lipase activity. C. cylindracea NRRL Y-17506 showed the highest lipase activity on all the typologies of OMW used. Its lipase production on OMW was markedly affected by the type of nitrogen source and was induced by the addition of olive oil. The highest activity (9.23 IU ml−1) of the yeast was obtained on OMW supplemented with NH4Cl (2.4 g l−1) and olive oil (3.0 g l−1).

The suitability of olive mill wastewater (OMW) as a growth medium for lipase production was assessed using Penicillium citrinum NRRL 1841, a versatile strain capable of producing lipase on several OMW typologies differing widely in their chemical oxygen demand, polyphenol and total sugar contents. Lipase production by P. citrinum in OMW-based media was significantly stimulated by nitrogen addition, with ammonium chloride proving to be the most effective source. In contrast, the addition of vegetable oils did not significantly affect lipase production. On the basis of these results, lipase production was subsequently optimised in shaken flasks by a central composite design. To this aim, the impact of three crucial variables, namely initial pH and concentrations of ammonium chloride and yeast extract, was investigated. Maximum lipase activity (ca 1230Udm −3 ) was reached after 188h of fermentation. The process was subsequently assessed in stirred tank and bubble column reactors of identical working capacity (3dm 3 ), leading to lower lipase production (735 and 430Udm −3 respectively) than in shaken flasks.  2006 Society of Chemical Industry

This paper presents a brief survey of studies conducted at the Laboratory of Applied and Environmental Microbiology of the University of Tuscia on the possible biotechnological valorisation of olive mill wastewater (OMW) using fungi. Besides being a serious environmental problem, OMW might be a possible resource owing to the presence of added value products (e.g. antioxidants) and of simple and complex sugars as a basis for fermentation processes. To this end the technical feasibility of various fungal fermentative processes either to obtain products of high added value or to improve its agronomic use has been assessed. With regard to the former aspect the following cases of study are described: production of enzymes, such as lipase by Candida cylindracea NRRL Y-17506, laccase and Mn-dependent peroxidase by Panus tigrinus CBS 577.79 and pectinases by Cryptococcus albidus var. albidus IMAT 4735, and exopolysaccharide production by Botryosphaeria rhodina DABAC-P82. As far as agronomic use of the waste is concerned, a process based on the acidogenic fungus Aspergillus niger NB2 and aimed at increasing the phosphorus content of OMW is also reported.  2006 Society of Chemical Industry

Monascus purpureus C322 was cultivated on well-established production media to yield prevailingly red or orange pigment-rich ethanolic extracts. Once these extracts had been diluted by an overall factor of 50, they were used as such to dye raw wool standard specimens differently premordanted using alum or stannic chloride. Independently of the mordant used, the specimens dyed with the red pigment-rich extracts showed a pale red colour tending to pink, whereas the specimens dyed with the orange pigment-rich extracts exhibited a more definite orange colour. By carrying out a few colourfastness standard tests (manual washing at 40 ◦ C, acid and basic perspiration and hot pressing), stannic chloride- premordanted wool specimens dyed with the red pigment-rich extracts were found to be less resistant to acid and basic perspiration than their orange counterparts. Since the production of the orange pigment- rich ethanolic extracts appeared to be more cost-effective than that of their red counterparts, the former might support the present demand for colorants of natural origin in the textile sector.  2005 Society of Chemical Industry

One-hundred and five fungal strains, belonging to 46 different species, were screened for exopolysaccharide production. Phytopathogenicity and, in particular, inability to produce conidia, were physiological characteristics positively associated and correlated with the fungal ability to produce polysaccharides. Among the 29 positive strains, Botryosphaeria rhodina DABAC-P82 was the most interesting reaching, when grown on optimal nitrogen source and concentration (NaNO3 and 2.0 g l(-1), respectively) and culture medium pH (3.7), 17.7 g l(-1) of exopolysaccharide production after only 24 h of fermentation; yield and productivity were 0.69 g g(-1) and 0.73 g l(-1)h(-1), respectively. The purified polysaccharide was characterised as a homopolysaccharide of glucose with a molecular weight of 4.875 x 10(6) Da. Studies of structural analysis indicated the presence of beta-1,3 and beta-1,6 linkages; the EPS structure was very similar to that of scleroglucan.

The filamentous fungus Phoma herbarum CCFEE 5080 isolated from continental Antarctica soil was tested for exopolysaccharide (EPS) production. The fungus grew and produced EPS (up to13.6 g/l) on a variety of carbon sources among which sorbitol was best, particularly at the concentration of 60 g/l. EPS production was maximum when the nitrogen source was NaNO 3 (3 g/l) and the incubation temperature was 28 °C. The polysaccharide was purified by repeated precipitation in ethanol and gel filtration and characterized as a homopolymer of glucose having a molecular weight of 7.412×10 6 ; structural analysis indicated the presence of β -1,3 and β -1,6 linkages only. After repeated freezing and thawing of the fungal biomass in the presence of EPS, the mycelial growth was much higher than that observed after freezing in the absence of EPS and the difference increased with the number of freeze–thaw cycles. It is hypothesized that the adaptation of P. herbarum CCFEE 5080 to the Antarctic soil microclimatic conditions, characterized by low temperature, high thermal fluctuations and repeated freeze–thaw cycles, might be related to the EPS production ability.

A jet-loop activated sludge reactor (JLR) of 15 dm3 working volume was used for the aerobic treatment of winery wastewater (WW). The reactor was operated continuously for more than 12 months using WW from different wineries and collected in different periods of the year with CODs that ranged between 0.8 and 12.8 kg m−3. Loading rates were from 0.4 to 5.9 kg-COD m−3 day−1 with hydraulic retention times that varied from 2.1 to 4.4 days. The system reacted well to sudden variations of loading: the COD removal efficiency was always higher than 90% with an organic load of the final effluents that ranged between 0.11 and 0.3 kg-COD m−3. After 185 days of continuous operation, the residential microbial population, of either the mixed liquor or the biofilm formed on the reactor's inner wall, was studied and characterized at the genus level. Most isolates belong to the genus Pseudomonas and the yeast Saccharomyces cerevisiae.

Ammonium fumarate production from glucose-based media by Rhizopus arrhizus NRRL 1526 with mycelial growth controlled by phosphorus limitation exhibited mixed-growth-associated product formation kinetics, with growth-associated production related to secondary mycelial growth only. The contribution of the primary mycelial growth phase was minimised by resorting to prolonged batch production using free mycelia under intermittent glucose feeding or repeated batch production using immobilised mycelia. The metabolic activity of free or immobilised mycelia was limited by fumarate accumulation or by oxygen diffusion phenomena, respectively. For batch cultures in a 15 dm3 stirred bioreactor the peripheral impeller speed (vI) was increased from 1.88 to 3.3 m s−1, and the fumarate yield coefficient on glucose increased from 0.25 ± 0.01 to 0.42 ± 0.02 g g−1, while the malate yield coefficient on fumarate (YM/F) reduced from 0.46 ± 0.01 to 0.14 ± 0.01 g g−1. With a net increase in the fumarate-to-malate ratio from 2 to 6.5, a vI value of 3.3 m s−1 gave the best fermentation performance and provided a basis for further scale-up studies. © 2002 Society of Chemical Industry

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, This work was financially supported by the Ministry of Science and Innovation of Spain (Project CTM2009-13140-C02-02)

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.

The production of itaconic acid from glucose-based media by Aspergillus terreus NRRL 1960 was found to be controlled by stirring rate and pH. When the phosphorous (P) level in the production medium was reduced to less than 10 mg l 1 , the fungal mycelium exhausted its primary growth and started to excrete itaconic acid, while it continued its secondary growth at the expense of ammoniacal nitrogen. The fermentation exhibited a mixed-growthassociated product formation kinetics, the non-growth associated production term (mI) being practically zero only when the pH was left free to change from 3.4 down to 1.85. On the contrary, when the pH was kept reducing up to a constant value by automatic addition of KOH 4 mol l 1 , the itaconate yield coefficient on the initial glucose supplied (YI:So) and mI and were 0.53 g g 1 and 0.028 h 1 at pH 2.4 and 320 rev min 1 and 0.5 g g 1 and 0.036 h 1 at pH 2.8 and 400 rev min 1 , respectively. Although the differences between mI and YI:So were statistically insignificant at the 95% confidence level, the net difference in the corresponding yield coefficients for itaconic acid on mycelial biomass resulted in a maximum itaconate production rate of 0.41 g l 1 h 1 at pH 2.8 and 400 rev min 1 , thus showing that this operating condition is no doubt optimal for the process under study. © 2000 Elsevier Science B.V. All rights reserved.

COD (chemical oxygen demand) removal rate and efficiency of winery wastewater (WW) aerobic treatments were evaluated in an air-bubble column bioreactor using self-adapted microbial populations either free or immobilized on polyurethane particles and in a packed-bed bioreactor immobilized on Raschig rings. The bioreactors were fed continuously for up to 12 months using WW of different origins and with different pollution loads (COD range, 0.8-11.0 kg.m(-3)): the maximum loading rate was approx. 8.8 kg-COD m(-3).d(-1). The highest COD removal rate (6.6 kg.m(-3).d(-1)) was obtained with free activated sludge in the bubble column bioreactor; treatment efficiency and hydraulic retention time were >90% and approx. 0.8 d, respectively. The microbial populations in the three reactors were characterized.

Direct ammonium fumarate production from glucose-based media with Rhizopus arrhizus NRRL 1526 was obtained using 2-kmol (NH4)2CO3 per-m3 as neutralising agent and controlling mycelial growth by phosphorous (P) limitation. As the P level in the production medium was increased from 0 to 0.3-kg of KH2PO4 per m3, the fumarate yield decreased from 0.32 to 0.13-g per-g of glucose consumed; maximum ammonium fumarate productivity (0.46-kg-m−3-h−1) was obtained when using 0.1-kg phosphate-m−3.

Effects of the polysaccharides alginate and locust bean gum, and oligosaccharides oligomannuronate (OM) and oligoguluronate (OG), on glucose oxidase (GOD) production by Penicillium variabile P16 were studied. Small increases were observed when the cultures were supplemented with OG and OM blocks with an average degree of polymerization (DP) of approximately ten. With 200 mg l−1 OM blocks addition at 0 h, the increase reached 32.1% compared with the control; however, regardless of the time of addition, large increases (up to approximately 70%) in GOD production were obtained with 100 and, particularly, 200 mg l−1 of alginate-derived oligosaccharides (OG and/or OM blocks) with a DP of approximately seven. No significant influence was observed on mycelial biomass.

Starchy materials such as corn starch, soft wheat flour, potato flour, cassava flour, sorghum starch, sweet potato and industrial potato flours, either acid or enzymatically hydrolysed, were used as substrates for itaconic acid production by Aspergillus terreus NRRL 1960. Both production and yield were highest on corn starch (18.4 gl - 1 and 34.0%, respectively). The degree of hydrolysis had a great influence on acid production which was highest when corn starch was saccharified at 85 DE (dextrose equivalent). In a 3 litre benchtop fermenter, itaconic acid production and productivity were 19.8 g l -1 and 0.13 g l -1 h -1 , respectively.

Mucor circinelloides LU M40 produced 12·2 mU ml−1 of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l−1 deionized water): pectin, 10·0; (NH4)2SO4, 1·0; KH2PO4, 2·0; Na2HPO4, 0·7; MgSO4.7H2O, 0·5; yeast extract, 1·0; Tween-80, 1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the Km value for linamarin was 2·93 mmol l−1. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.

Glucose oxidase production by the mutant M-80.10 and the parental strain of Penicillium variabile required the same nutrient addition to the production medium. Their corresponding batch cultures in a benchtop fermenter resulted in 25.6 and 19.1 U ml−1 of glucose oxidase activity, respectively, thus showing an overproduction of the mutant strain of about one third with respect to the wild type. In both strains, glucose oxidase production was not related to the autolysis phenomena, thus allowing the enzyme under study to be considered as extracellular.

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.

Aim of this work was to investigate the ability of Lentinus (Panus) tigrinus to degrade and detoxify a chlorobenzoate (CBA) mixture composed of mono-, di- and tri-chlorinated isomers. The degradation process was investigated as a function of both the growing medium ( i.e. low N Kirk's and malt extract-glucose medium) and cultivation conditions ( i.e. stationary and shaken cultures). The majority of CBAs were quantitatively degraded within the early 15 d from spiking with the notable exception of the double ortho -chlorinated compounds, 2,6-di-, 2,3,6-tri- and 2,4,6-tri-CBA. Analysis of the degradation intermediates indicated the occurrence of side chain reduction, hydroxylation and methylation reactions. Although CBAs stimulated laccase production, in vitro experiments with a purified L. tigrinus laccase isoenzyme demonstrated its inability to participate in the initial attack on CBAs even in the presence of redox mediators; similar results were found with a Mn-peroxidase isoenzyme. Conversely, prompt degradation was observed upon 1 h incubation of CBAs with a purified microsomal fraction containing cytochrome P-450 monooxygenase. The nature of some reaction products ( i.e. hydroxylated derivatives), the dependency of the reaction on NADPH and its susceptibility to either CO or piperonyl butoxide inhibition confirmed the involvement of L. tigrinus cytochrome P-450 in the early steps of CBA degradation.

High recalcitrant characteristics and low bioavailability rates due to aging processes can hinder high molecular weight polycyclic aromatic hydrocarbons (HMW-PAHs) bioremediation in real industrial polluted soils. With the aim of reducing the residual fraction of total petroleum hydrocarbons (TPH) and (HMW-PAHs) in creosote-contaminated soil remaining after a 180-d treatment in a pilot-scale biopile, either biostimulation (BS) of indigenous microbial populations with a lignocellulosic substrate (LS) or fungal bioaugmentation with two strains of white-rot fungi (WRF) (i.e., Trametes versicolor and Lentinus tigrinus) were comparatively tested. The impact of bivalent manganese ions and two mobilizing agents (MAs) (i.e., Soybean Oil and Brij 30) on the degradation performances of biostimulated and bioaugmented microcosms was also compared. The results reveal soil colonization by both WRF strains was clearly hampered by an active native soil microbiota. In fact, a proper enhancement of native microbiota by means of LS amendment promoted the highest biodegradation of HMW-PAHs, even of those with five aromatic rings after 60 days of treatment, but HMW-PAH-degrading bacteria were specifically inhibited when non-ionic surfactant Brij 30 was amended. Effects of bioaugmentation and other additives such as non-ionic surfactants on the degrading capability of autochthonous soil microbiota should be evaluated in polluted soils before scaling up the remediation process at field scale.

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.

Conidia ofPenicillium variabile P16 were immobilized in polyurethane sponge and used in repeated-batch processes in a fluidized-bed reactor. Optimal conditions for production of glucose oxidase and catalase were: inoculum size, 10%; glucose concentration, 80 g L−1; Ca-carbonate concentration, 15 g L−1; temperature, 28°C and aeration rate, 4 VV−1 min−1. In an extended repeated-batch process, glucose oxidase activity was highest after the fourth batch and catalase activity was highest after the fifth batch. Scanning electron microscopy showed that the fungus grew only in the interior of carrier particles.

Fumaric acid was produced in repeated batch processes using Rhizopus arrhizus NRRL 1526 immobilized in polyurethane sponge particles and cultivated in a fluidized-bed reactor. Fermentation conditions were established for concentration of carrier-particles and optimal concentrations of glucose nolasses (as glucose equivalent) and ammonium sulphate. Acid production was optimized (12·3 g/litre, 35·56% and 0·256 g/litre h of fumaric acid accumulation, yield and volumetric productivity of the whole process, respectively) under the following conditions: carrier-particle concentration, 20% v/v) of the working volume; glucose molasses, 50 g/litre; ammonium sulphate, alternation of cycles with 0·0 and 0·5 g/litre. The time of fermentation was 48 h per batch for a total of eight batches. Scanning electron microscopy revealed that the fungus began to grow within the particle cells but soon after the end of the first batch growth was essentially confined to the outer part of the polyurethane particle, where a peripheral layer of mycelium began to form.

The presence of antibiotic resistant bacteria was revealed among bacteria isolated from non carbonated mineral waters bottled in plastic (PVC) and in glass containers. Heterotrophic plate count values ranged between < 10 and 4.3 x 10(3) and between < 10 and 1.2 x 10(4) colony forming units/ml for the waters bottled in PVC and glass, respectively. The greatest resistance to a single antibiotic, 39.1% of 320 isolates from mineral waters, was found for nalidixic acid. Resistance to the other antibiotics was as follows: ampicillin (26.2%), bacitracin (19.7%), cotrimoxazole (18.7%), streptomycin (15.0%), tetracycline (14.4%), gentamycin (11.6%), chloramphenicol and rifampin (9.7%). The strains resistant to two or more antibiotics (multiple antibiotic resistant, MAR) provided 51% of the total isolates. Identification of 127 MAR strains showed that in the mineral waters gram-positive cocci dominated. The second, third and fourth group of identified MAR phenotypes were, in order to importance, gram-negative non-fermentative rods, gram-positive rods and gram-negative fermentative rods. The importance of the antibiotic resistant bacteria in mineral water is discussed.

Glucose oxidase (GOD) produced by a strain of Penicillium (Penicillium variabile P16) was employed, in a model system, to control the activities of tomato lipoxygenase (LPO), polyphenoloxidase (PPO), and peroxidase (POD). GOD in-hibited all three enzymes, at the typical pH of tomato fruit (4.3), i.e., the LPO was inhibited up to ca. 88%, the PPO up to ca. 63% and the POD up to ca. 50%. GOD also prevented β-carotene bleaching caused by these enzymes.

Conidia of Penicillium variabile P16 were subjected to mutagenesis and selection for glucose oxidase production on media containing o-dianisidine. Studies of the relationship between dose of UV irradiation and conidial survival and frequency of mutation showed that the best frequency of positive mutation (17%) was obtained in correspondence to a conidial survival of 52%. Out of 54 overproducing mutants tested in shaken flasks, M-80.10 showed the highest level of glucose oxidase activity (127% higher than the wild-type). M-80.10 mutant, transferred every 15 days to fresh medium and tested monthly for 8 months, appeared stable. The time course of growth and enzyme production by the mutant M-80.10 showed an increase of the glucose oxidase activity in the culture medium up to 19 U ml−1 after 96 h of fermentation.

Batch cultures of Penicillium variabile P16 were grown in a 3-1 benchtop bioreactor. Glucose oxidase and catalase production was followed while varying stirrer speed and adding buffering agents. Four hundred rev min−1 appeared to be optimum to ensure good growth and best enzyme production; above this, production was reduced. Among the different chemicals (CaCO3, NaOH, and NaOH plus CaCl2) employed to counteract the pH drop in the fermentation broth, CaCO3 gave the best results. The time course of growth, enzyme activities, and gluconate production at 400 rev min−1 and using CaCO3 as neutralizing agent is reported.

Aims To investigate the effect of support and growth medium (GM) on Pleurotus ostreatus biofilm production, specific metabolic activity (SMA) and ultrastructure. Methods and Results Biofilms were developed on membranes covering a broad range of surface properties and, due to the applicative implications of mixed biofilms, on standard bacterial GM in stationary and shaken culture. Hydrophilic (glass fibre, Duran glass and hydroxyapatite) and mild hydrophobic (polyurethane, stainless steel, polycarbonate, nylon) supports were more adequate for biofilm attachment than the hydrophobic Teflon. Among the GM, sucrose–asparagine (SA) was more conducive to biofilm production than Luria–Bertani and M9. GM was more influential than support type on biofilm ultrastructure, and a high compactness was evident in biofilms developed on SA. Biofilms on Duran glass were more efficient than planktonic cultures in olive-mill wastewater treatment. Conclusions The main effects of support and GM variables and their binary interactions on both biofilm production and SMA were all highly significant (P

Among biological approaches to the removal of aromatic amines and phenols from wastewater, the so-called enzyme-catalyzed polymerization and precipitation (ECPP) process relies on the use of oxidoreductases acting via radical mechanisms and characterized by a rather relaxed substrate specificity, such as laccase, tyrosinase and peroxidases. The main technical constraints of ECPP processes are due to a variety of enzyme deactivation phenomena occurring during catalysis and to the incomplete removal of oxidation products from solution. In order to put ECPP into practice, these drawbacks have to be either counteracted or minimized. Although several approaches, such as enzyme immobilization and reaction engineering, have been proposed to limit these constraints, this review is intended to provide a wide survey on some chemical additives with either protective or coagulating effects that have been so far employed for these purposes.

An industrial three-reactor plant treating 45 m(3) d(-1) of dairy wastewater was monitored to investigate the effect of different aeration regimes on performance efficiency and to find relationships with bacterial and protozoan communities in the activated sludge. During the study, the plant was maintained at six different "on/off" cycles of the blower (45/15, 15/15, 15/45, 30/30, 30/45 and 30/60 min), providing between 30.2 and 90.6 kg O(2) d(-1), and the main chemical/biochemical parameters (COD, BOD, NH(4)(+), NO(2)(-), NO(3)(-), PO(4)(3-), etc.) were determined. When at least 45.4 kg O(2) d(-1) (30/45) were provided, COD removal efficiencies were always in the range 88-94% but decreased to about 70% under aeration regimes 15/45 and 30/60. Ammonium ion degradation performance was compromised only in the lowest aeration regime (15/45). Total number of protozoa and their species richness, and bacterial viable counts and denaturing gradient gel electrophoresis (DGGE) profiles were used to characterize the microbiota of the activated sludge. Cell abundances and community structures of protozoa and bacteria were very similar in the three aerated reactors but changed with the aeration regimes. In particular, the 15/45 and 30/60 regimes led to low protozoan diversity with prevalence of flagellates of the genus Trepomonas at the expense of the mobile and sessile forms and, thus, to a less efficient activated sludge as indicated by Sludge Biotic Index values (3 and 4.5 for the two regimes, respectively). The structure of the bacterial community strongly changed when the aeration regimes varied, as indicated by the low similarity values between the DGGE profiles. On the contrary, number of viable bacteria and values of the biodiversity index remained stable throughout the whole experimentation. Taken together, the results of the present study clearly indicate that aeration regime variations strongly influence the structure of both protozoan and bacterial communities and, above all, that a high biodiversity among protozoan populations in the activated sludge is prerequisite for high performances in dairy wastewater treatment.

Background Several species belonging to the ecological group of white-rot basidiomycetes are able to bring about the remediation of matrices contaminated by a large variety of anthropic organic pollutants. Among them, polychlorobiphenyls (PCBs) are characterized by a high recalcitrance due to both their low bioavailability and the inability of natural microbial communities to degrade them at significant rates and extents. Objective of this study was to assess the impact of a maize stalk-immobilized Lentinus tigrinus CBS 577.79 inoculant combined with soybean oil (SO), as a possible PCB-mobilizing agent, on the bioremediation and resident microbiota of an actual Aroclor 1260 historically contaminated soil under unsaturated solid-phase conditions. Results Best overall PCB depletions (33.6 ± 0.3%) and dechlorination (23.2 ± 1.3%) were found after 60 d incubation in the absence of SO where, however, the fungus appeared to exert adverse effects on both the growth of biphenyl- and chlorobenzoate-degrading bacteria and the abundance of genes coding for both biphenyl dioxygenase (bph) and catechol-2,3-dioxygenase. A significant (P < 0.001) linear inverse relationship between depletion yields and degree of chlorination was observed in both augmented and control microcosms in the absence of SO; conversely, this negative correlation was not evident in SO-amended microcosms where the additive inhibited the biodegradation of low chlorinated congeners. The presence of SO, in fact, resulted in lower abundances of both biphenyl-degrading bacteria and bph. Conclusions The PCB depletion extents obtained in the presence of L. tigrinus are by far higher than those reported in other remediation studies conducted under unsaturated solid phase conditions on actual site soils historically contaminated by Aroclor 1260. These results suggest that the bioaugmentation strategy with the maize stalk-immobilized mycelium of this species might be promising in the reclamation of PCB-contaminated soils. The addition of SO to matrices contaminated by technical PCB mixtures, such as Aroclor 1242 and Delor 103 and characterized by a large preponderance of low chlorinated congeners, might not be advisable.

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.

Conidia of Penicillium variabile P16 producing glucose oxidase were immobilized in different carriers and used in repeated-batch processes. Limited free-cell growth and good mechanical stability of the carriers were obtained with Ca-alginale, agar and polyurethane sponge. During prolonged experiments, the polyurethane sponge appeared to be the best carrier in relation to glucose oxidase and catalase activities. The maximum mean volumetric productivity of gluconate was obtained using agar as immobilizing agent.

The ability of Lentinus tigrinus to grow and to degrade persistent aromatic hydrocarbons in aged contaminated soil was assessed in this study. L. tigrinus extensively colonized the soil; its degradation activity after 60 d incubation at 28 ◦C, however, was mostly limited to dichloroaniline isomers, polychlorinated benzenes and diphenyl ether while the fungus was unable to deplete 9,10-anthracenedione and 7-H-benz[DE]anthracene-7-one which were the major soil contaminants. Although clean-up levels were limited, both density of cultivable heterotrophic bacteria and richness of the resident bacterial community in L. tigrinus microcosms (LtM) increased over time to a significantly larger extent than the respective amended incubation controls (1.9×109 CFU g−1 vs. 1.0×109 CFU g−1 and 37 vs. 16, respectively). Naphthalene- and catechol 2,3-dioxygenase gene copy numbers, however, decreased over time at a higher rate in LtMthan in incubation controls likely due to a higher stimulation on heterotrophs than xenobiotics-degrading community members. L'articolo è disponibile sul sito dell'editore: http://www.sciencedirect.com

This article is a brief survey of the origin, characteristics, and possible reuses of various agriculture and agro-industrial wastes and wastewaters. Byproducts and effluents often contain substances of high-added value that can be directly recovered or can represent a zero- or low-cost substrate for fermentation processes aimed at producing biofuels or biomolecules of actual or potential commercial interest. At first, focus has been put on the general upgrading criteria and strategies. Then, a number of examples have been reported and examined concerning wastes and wastewaters such as residual agricultural and agro-industrial biomasses, relevant effluents (i.e., slaughterhouse, winery and starch industry wastewaters, and whey), and byproducts of the olive-oil extraction industry.

Glucose oxidase (GOD) production by 84 strains of the genus Penicillium was studied in order to define the distribution of the activity among and within the species. Plate screening and sub-screening in shake culture showed a rather homogeneous behaviour of strains belonging to the same species. Penicillium chrysogenum, P. expansum, P. italicum and P. variabile had a concomitant good production of GOD and catalase activity; peroxidase and polyphenol oxidase were never present. The capability of the culture filtrates to oxidase substrates other than glucose was also studied.

The effect of medium composition on glucose oxidase (GOD) production by the strain Penicillium variabile P16 was studied in shake culture. All the experiments were performed in a medium containing glucose and sodium nitrate as carbon and nitrogen sources, respectively. Addition of Fe 2+ to the medium was important to stimulate GOD production. Calcium carbonate was extremely important in maintaining the medium at a pH high enough so that the enzyme was not inactivated. The time course of growth and enzyme production by P. variabile P16 in the optimized medium is reported

This study comparatively investigated the PAH degradation ability of Lentinus tigrinus and Irpex lacteus in a historically polluted soil and creosote-impregnated shavings. With this regard, the effect of type of inoculum carrier (i.e., wheat straw, corn cobs and commercial pellets) and contaminant bioavailability was thoroughly determined. Although degradation performances of L. tigrinus were not significantly affected by the type of the support, they were invariably better than those of I. lacteus on both the polluted soil and the creosote-impregnated shavings. Although degradation efficiencies of all fungal microcosms were highly and significantly correlated with bioavailability, certain PAHs, such as chrysene and benzo[a]pyrene, were removed by L. tigrinus from the polluted soil at amounts that exceeded about 2.3-fold their respective bioavailabilities. Degradation of PAHs was negatively correlated with their organic carbon sorption coefficients (Koc) and hydrophobicity (log P). The strength of linear association with the latter parameter, however, was not affected by the type of contaminated matrix in L. tigrinus-based microcosms while it was significantly larger in the historically polluted soil than in the creosote-impregnated shavings in I. lacteus ones.

The ability of stationary and shaken Lentinus tigrinus CBS 577.79 liquid cultures to degrade a mixture of polycyclic aromatic hydrocarbons (PAHs) in N-rich (i.e., malt extract glucose, MEG) and in N-limited (low-N Kirk's medium, LNKM) media was investigated. Best results were obtained in shaken cultures where PAHs were degraded by 91% and 97% in MEG and LNKM, respectively; in stationary cultures, on the contrary, the degradation was never higher than 50%. Laccase activity was predominant on MEG while Mn-peroxidase (MnP) was preferentially produced in LNKM. The identification of degradation products showed the presence of several PAH derivatives, such as quinones, dicarboxylated and ring fission derivatives, presumably derived from the action of lignin-modifying enzymes. The presence of some degradation products (e.g., hydroxylated derivatives of anthrone and phenanthrene 9,10-dihydrodiol) suggested the possible involvement of cytochrome P-450-epoxide hydrolase system, the active form of which was found in 7-day-old cultures on MEG. In vitro experiments showed that the MnP from L. tigrinus had wider PAH substrate range and higher oxidation ability than the laccase produced by the same strain.

The possibility of using an endo-polygalacturonase produced by the yeast Cryptococcus albidus var albidus (yeast pectinase) in the mechanical olive oil extraction process and in the production of red wine was investigated. Compared with the control and olive pastes treated with a commercial enzyme preparation, an increase in oil yield was achieved by treatment with the yeast pectic preparation. Also, the finished oil quality (turbidity, oxidation induction time, chlorophyll, and the content of polyphenols and aromatic compounds) was generally better. Treatment of red musts with yeast pectinase resulted in an improved aromatic profile in the wines, even in the absence of an increase in colour intensity. Moreover, the use of the yeast pectinase did not cause any particular increase in the methanol content of the finished wines.

The objective of the study was to investigate the impact of chopped wheat straw (CWS), ground corn cobs (GCC) and commercial pellets (CP), as inoculum carriers, on both growth and polycyclic aromatic hydrocarbons (PAH) degradation performances of Dichomitus squalens, Pleurotus ostreatus and Coprinus comatus. A historically-contaminated soil (HCS) and creosote-treated shavings (CTS) from the Sobeslav wood preservation plant, characterized by different relative abundances of the PAH bioavailable fractions, were used to assess the contaminated matrix effect and its interaction with both carrier and fungal strain. In HCS, best results were obtained with CP-immobilized P. ostreatus, which was able to deplete benzo[a]anthracene, chrysene, benzo[b]fluoranthene (BbF), benzo[k]fluoranthene (BkF) and benzo[a]pyrene (BaP) by 69.1%, 29.7%, 39.7%, 32.8% and 85.2%, respectively. Only few high-molecular mass PAHs such as BbF, BkF and BaP were degraded beyond their respective bioavailable fractions and this effect was confined to a limited number of inoculants. In CTS, only phenanthrene degradation exceeded its respective bioavailability from 1.42 to 1.86-fold. Regardless of both inoculum carrier and fungal species, degradation was positively and significantly (P

A manganese peroxidase (MnP) isoenzyme from Panus tigrinus CBS 577.79 was produced in a benchtop stirred-tank reactor and purified to apparent homogeneity. The purification scheme involving ultrafiltration, affinity chromatography on concanavalin–A Sepharose, and gel filtration led to a purified MnP, termed ‘‘MnP II,’’ with a specific activity of 288 IU mg-1 protein and a final yield of 22%. The enzyme turned out to be a monomeric protein with molecular mass of 50.5 kDa, pI of 4.07, and an extent of N-glycosylation of about 5.3% of the high-mannose type. The temperature and pH optima for the formation of malonate manganic chelates were 45°C and 5.5, respectively. MnP II proved to be poorly thermostable at 50 and 60°C, with half-lives of 11 min and 105 s, respectively. Km values for H2O2 and Mn2+ were 16 and 124 lM, respectively. Although MnP II was able to oxidize veratryl alcohol and to catalyze the Mn2+ -independent oxidation of several phenols, it cannot be assigned to the versatile peroxidase family. As opposed to versatile peroxidase oxidation, veratryl alcohol oxidation required the simultaneous presence of H2O2 and Mn2+; in addition, low turnover numbers and Km values higher than 300 lM characterized the Mn2+ -independent oxidation of substituted phenols. Kinetic properties and the substrate specificity of the enzyme markedly differed from those reported for MnP isoenzymes produced by the reference strain P. tigrinus 8/18. To our knowledge, this study reports for the first time a thorough electrochemical characterization of a MnP from this fungus. L'articolo è disponibile sul sito dell'editore: http://www.springerlink.com

The potential use for agronomic purposes of dry olive-mill residue (DOR), solid waste from the olive oil two-phase extraction process, might be impaired by its phytotoxicity. Although fungal treatments can detoxify DOR, long times are required for these processes. The objective of this study was to assess whether the addition of immobilized fungal inocula to DOR might improve colonization rates, thus reducing the time necessary for its detoxification and bioconversion. Inocula of Panus tigrinus CBS 577.79 and Phlebia sp. DABAC 9 immobilized on either chopped maize stalks or polyurethane sponge (PS) led to higher removals of both phenols and phytotoxicity from DOR than free inocula after 4 weeks of incubation. Best dephenolization (85%) was with PS-immobilized Phlebia sp., the use of which reduced germinability inhibition of Lepidium sativum and Lactuca sativa by 80 and 71.4%, respectively. Regardless of the type of inoculant, a low degree of humification was obtained.

Fumaric acid production by Rhizopus arrhizus from potato flour was studied at different initial substrate concentrations (S), C/N ratios and fermentation times (t f) in a composite design experiment. By using response surface methodology and canonical analysis, the experimental values of fumaric acid and mycelial biomass yields and productivity were fitted to the only statistically significant factors with mean percentage errors of 11, 26 and 13%, respectively. Owing to the limited statistical significance of the C/N ratio, it was possible to determine the values of S (25–50 g/l of glucose equivalent) and t f(44–100 h) associated with fumaric acid yields ranging from 60 to 75% and productivities varying from 6 to 8.4 g/l per day. Since such results were in quite good agreement with previous experiments carried out on other starchy materials, the above operating conditions might be used to minimize fumaric acid production costs as a function of the feed-stock used.

The morphological development of a glucoamylase-producing strain of Aureobasidium pullulans immobilized in Ca-alginate was investigated during semicontinuous fermentations in a fluidized-bed reactor. A dense mycelium layer was developed by the fungus on the gel bead surface which gave the beads a "pellet-like" configuration. Varying culture conditions, such as bead, substrate and CaCl2 concentrations and aeration rate, had a consistent effect on both enzyme production and morphological development of the "in situ" grown cells. Optimization of the growth conditions appeared to be necessary to prevent fungal overgrowth and to enable the immobilized inoculum to continue a sustained level of glucoamylase production over a great number of transfers.

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)).

Yeast-like cells ofAureobasidium pullulans were immobilized in Ca-alginate gel beads and employed for continuous production of glucoamylase in a fluidized-bed reactor (250 ml working volume). After an activation time of 48 h, to allow the “in situ” germination of the fungal blastospores, the reactor was operated continuously for over 150 h. A steady state enzyme concentration of 1.2–1.3 U ml−1 of glucoamylase activity and an enzyme volumetric productivity of ca. 130 U ml−1 h−1 were obtained at a medium flow rate of 26 ml h−1. Enzyme activity and volumetric productivity were influenced by fermentation conditions such as inoculum size and airflow rate.

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 x 10(8) and 8.35 +/- 0.6 x 10(8), respectively, vs 9.2 +/- 0.3 x 10(7)). 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.

Aims: Characterization of β-glucan production from Botryosphaeria rhodina DABAC-P82 by detecting simultaneously glucan-hydrolytic enzymes and their localization, culture medium rheology and oxygen transfer. Methods and Results: Mycelium growth, β-glucan production, substrate consumption and glucan-hydrolytic enzymes were monitored both in shaken flasks and in a 3-l stirred-tank bioreactor. Glucan production (19·7 and 15·2 g l−1, in flask and bioreactor, respectively) was accompanied by extra-cellular and cell-bound β-glucanase and β-glucosidase activities. In the bioreactor scale, in the time interval of 0–78 h the apparent viscosity of the culture broth exhibited a general increase; thereafter, it began to reduce, probably because of the above glucan-hydrolytic activities. Moreover, the culture media collected after 45 h behaved as solid-like materials at shear rates smaller than 0·001 s−1, as pseudo-plastic liquids in the middle shear rate range and as Newtonian ones at shear rates greater than 1000 s−1. Conclusion: The greatest β-glucan accumulation in the bioreactor was found to be associated with nitrogen and dissolved oxygen concentrations smaller than 0·15 g l−1 and 25%, respectively, and with the peak points of the glucan-degrading enzymes. Significance and Impact of the Study: A careful analysis of the critical factors (such as, culture broth rheology, oxygen mass transfer and glucan-hydrolytic enzymes) limiting the β-glucan production by B. rhodina is a prerequisite to maximize β-glucan yield and production, as well as to define the process flow sheet capable of maximizing biopolymer recovery, solvent re-utilization and glucose consumption.