DIBAF

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±0.3×10 8 and 8.35± 0.6×10 8 , respectively, vs 9.2±0.3×10 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.

Contaminated soil from a historical industrial site and containing sulfide ore ashes and aromatic hydrocarbons underwent sequential leaching by 0.5 M citrate and microbial treatments. Heavy metals leaching was with the following efficiency scale: Cu (58.7%) > Pb (55.1%) > Zn (44.5%) > Cd (42.9%) > Cr (26.4%) > Ni (17.7%) > Co (14.0%) > As (12.4%) > Fe (5.3%) > Hg (1.1%) and was accompanied by concomitant removal of organic contaminants (about 13%). Leached metals were concentrated into an iron gel, produced during ferric citrate fermentation by the metal-resistant strain BAS-10 of Klebsiella oxytoca. Concomitantly, the acidic leached soil was bioaugmented with Allescheriella sp. DABAC 1, Stachybotrys sp. DABAC 3, Phlebia sp. DABAC 9, Pleurotus pulmonarius CBS 664.97, and Botryosphaeria rhodina DABAC P82. B. rhodina was most effective, leading to a significant depletion of the most abundant contaminants, including 7-H-benz[DE]anthracene-7-one, 9,10-anthracene dione and dichloroaniline isomers, and to a marked detoxification as assessed by the mortality test with the Collembola Folsomia candida Willem. The overall degradation activities of B. rhodina and P. pulmonarius appeared to be significantly enhanced by the preliminary metal removal.

The impact of several mobilizing agents (MAs) (i.e., soybean oil, Tween-20, Tween-80, olive-oil mill wastewaters, and randomly methylated b-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 À1 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 Â 10 7 CFU g À1 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 b-cyclodextrins (39 vs. 33 and 1.43 vs. 1.26, respectively) and soybean oil (19 vs. 5 and 1.01 vs. 0.65, respectively).

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 × 10 9 CFU g −1 vs. 1.0 × 10 9 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 LtM than in incubation controls likely due to a higher stimulation on heterotrophs than xenobiotics-degrading community members.

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.

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 288C. 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-2,3dioxygenase (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 2.8and 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-, octaand 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.

Industrial three-reactor plant Different aeration regimes Microbial community structure Protozoa Bacterial PCR-DGGE a b s t r a c t An industrial three-reactor plant treating 45 m 3 d À1 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 À1 , and the main chemical/biochemical parameters (COD, BOD, NH þ 4 , NO À 2 , NO À 3 , PO 3À 4 , etc.) were determined. When at least 45.4 kg O 2 d À1 (30/45) were provided, COD removal efficiencies were always in the range 88e94% 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. ª (M. Petruccioli).

Direct ammonium fumarate production from glucose-based media with Rhizopus arrhizus NRRL 1526 was obtained using 2 kmol (NH 4 ) 2 CO 3 per m 3 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 KH 2 PO 4 per m 3 , 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 .

Nine fungal strains isolated from an aged and heavily contaminated soil were identified and screened to assess their degradative potential. Among them, Allescheriella sp. strain DABAC 1, Stachybotrys sp. strain DABAC 3, and Phlebia sp. strain DABAC 9 were selected for remediation trials on the basis of Poly R-478 decolorization associated with lignin-modifying enzyme (LME) production. These autochthonous fungi were tested for the abilities to grow under nonsterile conditions and to degrade various aromatic hydrocarbons in the same contaminated soil. After 30 days, fungal colonization was clearly visible and was confirmed by ergosterol determination. In spite of subalkaline pH conditions and the presence of heavy metals, the autochthonous fungi produced laccase and Mn and lignin peroxidases. No LME activities were detected in control microcosms. All of the isolates led to a marked removal of naphthalene, dichloroaniline isomers, o-hydroxybiphenyl, and 1,1-binaphthalene. Stachybotrys sp. strain DABAC 3 was the most effective isolate due to its ability to partially deplete the predominant contaminants 9,10-anthracenedione and 7H-benz[DE]anthracen-7-one. A release of chloride ions was observed in soil treated with either Allescheriella sp. strain DABAC 1 or Stachybotrys sp. strain DABAC 3, suggesting the occurrence of oxidative dehalogenation. The autochthonous fungi led to a significant decrease in soil toxicity, as assessed by both the Lepidium sativum L. germination test and the Collembola mortality test.

Fumaric acid production by Rhizopus arrhizus from potato flour was studied at different initial substrate concentrations (S), C / N ratios and fermentation times (tF) 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/1 of glucose equivalent) and tv (44-100 h) associated with fumaric acid yields ranging from 60 to 75% and productivities varying from 6 to 8.4 g/1 per day. Since such resuits were in quite good agreement with previous experiments carried out on other starchy materials, the above operating conditions might be used to minimise fumaric acid production costs as a function of the feedstock used.

Cyclic fed-batch plus batch polygalacturonase production by Aureobasidium pullulans in slurry fermentation systems using raw orange peel as substrate was studied in a 3-dm 3~ stirred fermentor by setting the main operating variables ( T = 297 ° K; pHo = 3.2; OPo=3% w / v ; n = 7 0 0 r p m ) to optimal values determined previously. In this way, it was possible to stabilize enzyme excretion at 130-140 VU cm -3. The time course of this fermentation process in terms of cell growth, substrate consumption and enzyme synthesis was reconstructed with a mean standard error less than 10%, by applying an unstructured model set up in a batch run and further refined in a series of cyclic fedbatch plus batch operations. In particular, the enzyme formation rate was related to the effect of reducing sugars as inhibitors at higher concentrations and as activators at lower levels by using an exponential equation. Moreover, the consumption rate of reducing sugars was found to be linearly related to the cell growth rate, its specific rate being of pseudo-first order with respect to the reducing sugar concentration.

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 aVected 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 NH 4 Cl (2.4 g l ¡1 ) and olive oil (3.0 g l ¡1 ).

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 À1 NH 4 Cl and 3 g l À1 olive oil led to an enzyme activity of about 10 U ml À1 . 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 À1 ) when the pH was held constant at 6.5, significantly increased (18.7 U ml À1 ) with uncontrolled pH and was maximum (20.4 U ml À1 ) 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 À1 ) and mean volumetric productivity (i.e., 123.5 U l À1 h À1 ).

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 À 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.

Phanerochaete chrysosporium NRRL 6361 and Pleurotus pulmonarius CBS 664.97 were tested for their ability to grow under nonsterile conditions and to degrade various aromatic hydrocarbons in an aged contaminated soil that also contained high concentrations of heavy metals. After 24 days fungal incubation, carbon-CO 2 liberated, an indicator of microbial activity, reached a plateau. At the end of the incubation time (30 days), fungal colonization was clearly visible and was confirmed by ergosterol and cell organic carbon determinations. In spite of unfavorable pH (around 7.4) and the presence of heavy metals, both fungi produced Mn-peroxidase activity. In contrast, laccase and aryl-alcohol oxidase were detected only in the soil treated with P. pulmonarius CBS 664.97 and lignin-peroxidase in that with P. chrysosporium NRRL 6361. No ligninmodifying enzyme activities were present in non-inoculated soil incubated for 30 days (control microcosm). Regardless of the fungus employed, a total removal of naphtalene, tetrachlorobenzene, and dichloroaniline isomers, diphenylether and N-phenyl-1-naphtalenamine, was observed. Significant release of chloride ions was also observed in fungal-treated soil, in comparison with that recorded in the control microcosm. Both fungi led to a significant decrease in soil toxicity, as assessed using two different soil contact assays, including the Lepidium sativum L. germination test and the Collembola mortality test.

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 Soběslav 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 < 0.001) correlated with bioavailability in fungal microcosms on HCS and CTS and such correlation was very similar in the two matrices (R 2 adj equal to 0.60 and 0.59, respectively). The ability of white-rot fungi to degrade certain PAHs beyond their bioavailability was experimentally proven by this study. Although CTS and HCS considerably differed in their physico-chemical properties, PAH contents and contaminant aging, the relationship between degradation and bioavailability was not significantly affected by the type of matrix.