Maurizio Petruccioli

Role of Autochthonous Filamentous Fungi in Bioremediation of a Soil Historically Contaminated with Aromatic Hydrocarbons

ABSTRACT 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.

Aerobic treatment of winery wastewater using a jet-loop activated sludge reactor

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 reactors inner wall, was studied and characterized at the genus level. Most isolates belong to the genus Pseudomonas and the yeast Saccharomyces cerevisiae.

Production and structural characterization of the exopolysaccharide of the Antarctic fungus Phoma herbarum CCFEE 5080

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 to 13.6 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 NaNO3 (3 g/l) and the incubation temperature was 28 degrees 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 x 10(6); structural analysis indicated the presence of beta-1,3 and beta-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.

High-rate aerobic treatment of winery wastewater using bioreactors with free and immobilized activated sludge.

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.

Comparative assessment of bioremediation approaches to highly recalcitrant PAH degradation in a real industrial polluted soil

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.

Effect of pH and stirring rate on itaconate production by Aspergillus terreus

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-growth-associated 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 (Y(I/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 Y(I/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.

Enhancement of glucose oxidase production by Penicillium variabile P16

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.

Effect of stirrer speed and buffering agents on the production of glucose oxidase and catalase by Penicillium variabile (P16) in benchtop bioreactor

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.

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

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

In vivo and in vitro polycyclic aromatic hydrocarbons degradation by lentinus (panus) tigrinus CBS 577.79.

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 Kirks 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.