Feasibility of Solid‐State Fermentation Using Spent Fungi‐Substrate in the Biodegradation of PAHs

The ability of Trametes versicolor and Pleurotus ostreatus to grow on agroindustrial wastes, such as orange peels, and to degrade model polycyclic aromatic hydrocarbons (PAHs) in solid‐state fermentation (SSF) was evaluated. Best results in laccase production were obtained with T. versicolor cultures (3000 U L^?1), however, P. ostreatus (2700 U L^?1) showed higher ability to degrade the tested PAHs phenanthrene (PHE) and pyrene (PYR). Moreover, orange peels can be used as biofertilizer and the oxidative ability of the spent fungus and substrate obtained in SSF can be used in the remediation of polluted soil. The results obtained confirm this hypothesis. Thus, the decontamination achieved in soils polluted with PYR was around 43% (after 30 days) by addition of spent fungus and substrate from SSF.     

Production of Laccase from Coriolus versicolor and Its Application in Dye Decolorization in Combination with UV/H2O2 Technique

The relative ability of Coriolus versicolor to grow on coir fiber as a ligninocellulosic material was examined. Addition of yeast extract to the culture increased laccase activity, which was further enhanced to the level of 1976 U/L by addition of 1 mM copper sulfate. Laccase thus produced was used without further purification for the decolorization of various dye solutions. Decolorization efficiency was compared with the conventional environment friendly oxidation technique using hydrogen peroxide in the presence of UV radiations. Laccase showed good decolorization in most of the cases. Excellent results were achieved when the dye solution was treated successively with laccase and UV/H₂O₂ wherein more than 80% decolorization was achieved. This value is remarkably higher than that attained either by the enzyme or UV/H₂O₂ photolysis alone.     

Efficiency of Pleurotus florida Laccase on Decolorization and Detoxification of the Reactive Dye Remazol Brilliant Blue R (RBBR) under Optimized Conditions

Laccase from the white‐rot fungus Pleurotus florida, produced under solid‐state fermentation conditions, was used for the decolorization of reactive dye Remazol Brilliant Blue R (RBBR). RBBR was decolorized up to 46% by P. florida laccase alone in 10 min. In the presence of N‐hydroxybenzotriazole (HBT), the rate of decolorization was enhanced 1.56‐fold. Central composite design of response surface methodology with four variables namely, dye, enzyme, redox mediator concentrations, and time at five levels was applied to optimize the RBBR decolorization. The predicted optimum level of variables for maximum RBBR decolorization (87%) was found to be 52.90 mg L^?1 (RBBR), 1.87 U mL^?1 (laccase), 0.85 mM (HBT), and 7.17 min (time), respectively. The validation results showed that the experimental value of RBBR decolorization (82%) was close to the predicted one. The disappearance of C–N and C–X groups, and a small shift in N–H groups in Fourier‐transform infra red (FTIR) spectroscopy confirms the degradation of RBBR chromophore by laccase enzyme. The phytotoxicity of RBBR was considerably reduced after the treatment with laccase. RBBR decolorization kinetics; K_m and V_max were calculated to be 145.82 mg L^?1 and 24.86 mg L^?1 min, respectively.     

Formation of Humic-like Substances in Mixed and Pure Cultures of Aquatic Microorganisms

A natural decay of plant and animal biomass in aquatic environments results in the release of different simple structured organic substances into water. In our laboratory experiments we investigated whether some of them, e.g., starch or peptone, and also a complex yeast biomass can be transformed into humic-like substances by natural assemblages and some pure cultures of aquatic microorganisms. After a 6 months incubation most of the cultural media enriched with those natural organic substrates turned dark in color and humic-like substances (HS) could be isolated. However, the original substrate organic carbon was mainly mineralized in microbial cultures, and only about 3 % C was converted into HS. Total yields of HS differed in dependence of the individual substrate used (peptone > yeast > starch), the origin of inoculum (river > lake > groundwater), and the incubation temperature (20°C > 10°C). According to their elemental composition, and their spectroscopical and electrophoretic characteristics the microbially formed HS resembled natural aquatic humic matter, but were higher in aliphatic constituents (carbohydrates, peptides) and lower in aromatic structures. HS-like substances were also obtained from pure cultures of a bacterium Bacillus sphaericus and a fungus Cladosporium cladosporioides. The ¹³C and ¹⁵N isotopic contents of the microbially produced HS differed in dependence on the microbial inoculum and the type of organic substrates used, but in general they indicated anabolic processes to be mainly responsible for the humification of the simple organic substrates used in our experiments.     

Dissolved Humic Substances Can Directly Affect Freshwater Organisms

Direct effects of pure humic substance (BS1 FA) on three different aquatic organisms (Ceratophyllum demersum, Dreissena polymorpha, and Chaetogammarus ischnus) were demonstrated in this study. Exposure to environmentally relevant concentrations (0.5 mg/L) of this humic substance led to the activation of the microsomal and soluble glutathione S‐transferase (GST) in Ceratophyllum demersum. Exposure to 3‐chlorobiphenyl showed also an elevation of GST activity, which is due to the proposed detoxication metabolism of these chlorinated biphenyl. Coexposure to a mixture of 3‐chlorobiphenyl and BS1 FA showed a reduction of enzyme elevation, but still significant over an untreated control. The direct impact of humic substances seems not to be restricted to a specific class of organisms.     

Tracing of Peroxidase Activity in Humic Lake Water

An enzyme assay was developed for studies on peroxidase activities in humic lake water. 3,4-Dimethoxybenzyl alcohol (veratryl alcohol, VeraOH) was used as tracer substrate, and peroxidase (EC 1.11.1.7) activity was measured by high-performance liquid chromatography. The chemical stability of VeraOH and its application as peroxidase substrate was tested under light and dark conditions, different hydrogen peroxide (H₂O₂) concentrations and humic matter contents. VeraOH was stable under low UV radiation at in situ conditions in lake water (<0.010...0.25 kJ m^–2 d^–1), laboratory conditions (<0.05...0.30 kJ m^–2 d^–1), and low (1...100 μM) H₂O₂ concentrations. However, peroxides oxidized VeraOH above 1...10 mM H₂O₂ concentration in sterile Millipore-Q and humic lake water. Dark incubations showed little VeraOH oxidation products. The developed peroxidase assay was tested in the growth medium of Phanerochaete chrysosporium and a bacteria isolate (P.M.D. 20.4.3.1) from mesohumic lake Pääjärvi. Peroxidase activities were also measured in natural microbial communities under standard laboratory and under in situ conditions in humic lake water. Incubation times of about 5 to 12 days were usually needed to record significant (P < 0.05) peroxidase activities, in lake waters. In situ peroxidase activities varied in pelagial surface water (0...0.5 m) on a seasonal scale between 74 nmol L^–1 h^–1 and 273 nmol L^–1 (mean: 176 nmol L^–1 h^–1) and within the water column between 110 nmol L^–1 h^–1 and 800 nmol L^–1 h^–1 (mean: 500 nmol L^–1 h^–1) in polyhumic lake Mekkojärvi.     

Organic matter,sediment particle size and nutrient distributions in a sewage affected shallow channel

Sediment and seawater samples were taken in a sewage affected channel in Blanca Bay, Argentina. Protein content, ‘humic substances’ and silt-clay percentage were evaluated in the sediments; NH₄⁺, PO₄^3? and ‘dissolved organic matter’ in seawater. A highly significant correlation (r=0.747, n=59) was obtained for the relation between protein and % silt-clay. The ‘humic substances’ distribution depends more on the distance from the outfall than does the protein content, probably because of the important relation of the latter with the sediment particle size. The dissolved components showed a dilution pattern similar to that of a contaminant plume in a moving water mass, reaching normal values at 1700 m from the outfall.     

水华微囊藻(Microcystis flos-aquae)生长周期中腐殖酸的释放特性

由于严重的水体富营养化,太湖长期暴发蓝藻水华,这些蓝藻在代谢及消亡过程中大量释放包括腐植酸在内的有机物,不仅影响供水水质,并且进一步加重了水华程度.以太湖水华优势藻种——水华微囊藻(Microcystis flos-aquae T34)为研究对象,对藻株生长周期中的藻细胞、浮游细菌进行计数,并使用TOC仪与三维荧光光谱对培养液中提取的腐殖酸进行定量与定性分析,探究水华微囊藻在生长过程中藻细胞密度、浮游细菌密度与腐殖酸浓度的变化规律.结果表明,培养液中腐殖酸浓度的变化趋势与细菌数量变化趋势一致,但与细菌数量曲线拐点相比,腐殖酸浓度曲线拐点出现两周的延迟.腐殖酸产量在藻细胞对数期较低,当水华微囊藻进入稳定期与衰亡期后,腐殖酸大量产生,其浓度迅速增加,最高可达到28.6 mg/L.根据三维荧光光谱分析,水华微囊藻所产生的腐殖酸特征峰出现在(235~245 nm)/(380~425 nm),属于类富里酸荧光峰.本研究初步探究蓝藻与水体腐殖酸之间的关系,为水体腐殖酸来源的研究提供了新的思路.     

Photochemical activity and fluorescence of electrophoretic fractions of aquatic humic matter

A combination of sephadex gel-chromatography and polyacrylamide gel electrophoresis was used to obtain stable electrophoretic fractions with different molecular sizes from a preparation of humic substances isolated from river water by reverse osmosis. The photochemical activity of humic substances and their fractions was determined. For the first time it is shown that low-molecular fractions of aquatic humic substances, when irradiated by polychromatic light, can intensely fluoresce and initiate the formation of singlet O₂, effectively destroying model compound furfural. The photochemical activity and intensity of fluorescence of high-molecular fractions is insignificant. The obtained data will help to extend the current knowledge in the field of natural water hydrochemistry about the ways of its natural self-purification from anthropogenic pollutants.     

Spectroscopic Characterization of Organic Structures and Organic‐Inorganic Interactions in Paper Mill Sludge

The organic composition and organic‐inorganic interaction in paper mill sludge (PS) solvent extracts (hexane, ethyl acetate, acetone and ethanol) and humic fractions, humic acid (HA) and humin (HU) were studied by electron paramagnetic resonance spectroscopy (EPR), proton and carbon‐13 nuclear magnetic resonance spectroscopy (¹H NMR; ¹³C NMR), Fourier‐transformed infrared spectroscopy (FTIR), and ultraviolet‐visible spectroscopy (UV‐vis). The strategy of fractionating the PS, sequentially, with organic solvents of increasing polarity is a reliable analytical procedure for humic substance sample separation because it results in more purified fractions. FTIR, ¹H NMR and ¹³C NMR results showed that hexane extract consisted mainly of aliphatic hydrocarbon structures. Their contents in the extracts decreased as the polarity of the extracting solvent increased and the content of oxygen functional groups increased. Carboxylic and carboxylate functional groups were found in the acetone extract, and ester and ether functions were predominantly found in the ethanol extract. EPR spectra revealed some Fe³⁺ complexes with rhombic structure (g₁ = 4.3; g₂ = 9.0) in the humic fractions and in all solvent extracts, except hexane. Quasi‐octahedral Fe³⁺ complexes (g = 2.3; ΔH_pp ≤ 400 G) were found in the HU fraction and in the acetone extract. The organic free radical content in the HA fraction was higher than the non‐fractionated PS sample and HU fraction.     

Spectroscopic investigation of humic substances in a tropical lake during a complete hydrological cycle

Fluorescence and UV‐VIS techniques were employed for the investigation of natural organic matter (NOM) of a tropical lake. The relationships of absorbance/dissolved organic carbon (A/DOC), fluorescence intensity/dissolved organic carbon (FI/DOC), fluorescence ratio (FR), and peak wavelength with the highest intensity (PW) were used to distinguish the pedogenic or aquagenic origin of NOM. The values of FR, PW and A₂₈₅/DOC of high waters (HW) or flooded period samples and of low waters (LW) period samples of the dry season, except for September 2002, confirm the predominance of pedogenic material. The spectra of water were similar to the standard fulvic acid (FA), and the spectra of FA from the lake were similar to the nearby soils, indicative of pedogenic predominance. The results confirm that the dissolved NOM of Patos Lagoon – MS (Brazil), in all sampling periods, predominantly consisted of humic substances (FA) of pedogenic origin.     

Ageing of Dissolved Halogenated Humic Substances and the Microbiological Influence on this Process

The distribution of halogens in various fractions of humic substances (HS), separated by their molecular weight, was found to be different for the different halogens. This was demonstrated for chlorine, bromine, and iodine in sewage and brown water samples by applying inductively coupled plasma mass spectrometry coupled with size‐exclusion chromatography. Quantification of the different fractions of iodinated humic substances was obtained by quadrupole mass spectrometry in connection with the isotope dilution technique using an ¹²⁹I‐enriched spike solution. Quantitative analysis was not possible for the corresponding chlorine and bromine species because of spectrometric interferences in the quadrupole instrument. The ageing of HS/halogen species was followed with respect to possible transformations of these species in a ground and sewage water sample up to eight weeks. Even if a distinct structural variation of the humic substance was observed with time by measuring the UV absorption, chlorine remained in the same molecular weight fraction and only a small change was found for the HS/bromine species after eight weeks. In contrast to these findings a substantial transformation of HS/iodine compounds took place, which demonstrated that the transfer probability of halogens from one to another HS fraction is increased with decreasing strength of the halogen bond to carbon. By comparing the results of an original sewage water sample with a filtered one and with another one which was enriched by microorganisms cultivated from the same original sample, a strong microbiological influence on the transformation of HS/iodine species was found. A quantitative balance of the corresponding HS/iodine fractions was calculated for an ageing period of eight weeks showing that iodine was preferably transferred to newly formed UV active HS substances of high molecular weight. In total, no iodine was released from the humic substances.     

Interaction of Aquatic Dissolved Organic Matter(DOM) with Amitrole: The Nature of the Bound Residues

In this study the binding behaviour of the herbicide amitrole with humic substances respectively dissolved organic matter (DOM) is presented. The results of the binding and release experiments using radio actively ¹⁴C-labelled amitrole ledto the conclusion that the binding of amitrole is very low. About 70% of amitrole bound to humic substances was not extractable. The releasable amitrole portion of the purified DOM-amitrolecomplexes led to a harmful effect in the cress test. Solid-state cross polarisation (CP) magic angle spinning (MAS) ¹⁵N-NMR spectroscopy is a powerful spectroscopic method to elucidate the structure of bound xenobiotics. In addition extraction experiments were used resulting in a binding scheme of amitrole to DOM, where above all the covalent bonding occurred via acid amide bonds.     

Application of Hydrophobic Interaction Chromatography (HIC) in Water Analysis Anwendung der Hydrophobizitätschromatographie (HIC) in der Wasseranalytik

Dissolved high molecular weight organic substances, like humic substances, can be characterized with regard to their molecular weight distribution using gel filtration chromatography (GFC). In addition, the adsorption properties, for example on activated carbon, can be described, if the substances are separated by hydrophobic interaction chromatography (HIC) in different fractions using a chromatographic column with a hydrophobic matrix. The retention time (Rt) of single chemical substances in this column can be used to characterize the hydrophobicity or hydrophility of each compound as well as of the high molecular weight organic matter. The hydrophobic interaction between the matrix of the column and the dissolved chemical compounds also depends on the hydrophobicity of the gel and the composition of the eluent. Relations between retention times, log P-coefficients (octanol/water partition) and K-values of isotherms on activated carbon (Freundlich equation) were found.     

过量Zn2+对水花生(Alternanthera philoxeroides)愈伤组织氧化胁迫及多胺代谢的影响

以植物组织培养技术培养的水花生愈伤组织为实验材料,研究了过量Zn2+(0、0.2、0.4、0.8、1.6 mmol/L)对水花生愈伤组织的氧化损伤及多胺代谢的影响.结果表明,随着Zn2+浓度增加:(1)水花生愈伤组织的超氧阴离子产生速率和硫代巴比妥酸反应物水平显著提升,超氧化物歧化酶活性明显抑制,叶绿素a含量显著降低;(2)精氨酸脱羧酶和鸟氨酸脱羧酶活性均升高,多胺氧化酶和二胺氧化酶活性在低Zn2+浓度处升高、高Zn2+浓度处降低;(3)总腐胺和总精胺含量在低Zn2+浓度处降低、高Zn2+浓度处增加,总亚精胺含量仅在Zn2+浓度为1.6 mmol/L时有显著增加;(4)游离态腐胺含量变化与总腐胺含量相似,游离态亚精胺含量在高Zn2+浓度时下降,游离态精胺含量仅在Zn2+浓度为0.2 mmol/L时略有下降;(5)结合态腐胺和精胺在Zn2+浓度为0.2 mmol/L时下降而在其余处理组中上升,结合态亚精胺含量各处理组中均增加;(6)束缚态腐胺和亚精胺含量均下降.可见,过量Zn2+胁迫导致水花生愈伤组织氧化性损伤,并扰乱其多胺代谢平衡;束缚态多胺向结合态多胺的转化是过量Zn2+胁迫下水花生愈伤组织多胺代谢变化的一个显著特征;以结合态为代表的内源性多胺积累对水花生愈伤组织抵抗过量Zn2+胁迫具有重要意义.     

Reconstructing the development of a gully in the Upper Kalaus basin,Stavropol region (southern Russia)

The Stavropol region of southern Russia is severely affected by human‐induced gully erosion. A lack of detailed information on the different stages of gully formation resulting from major agricultural expansion c. 100 years ago, is an obstacle for management and containment of these systems. In this study we combine measurements of particle‐bound radionuclides (¹³⁷Cs, ²¹⁰Pb_ex, ²²⁶Ra, ²³²Th and ⁴⁰K) and classical geomorphology to investigate and reconstruct the different phases of development of a gully during the last c. 100 years. We believe the ?rst phase (1) involved an initial incision into the bottom of a small valley (catchment area c. 1 km²) about 100 years ago. A short period of rapid growth was followed by a longer stage of gully stabilization. Subsequent phases were: (2) the period 1954–1960 – re‐incision in the lower gully reach was initiated by a high‐magnitude rainfall event, and a substantial amount of sediment was delivered to the gully fan; (3) c. 1960–1986 – the knickpoint retreated slowly, sediment was redeposited nearby, and the fan surface became stable; (4) 1986–1987 – a dam was built in the gully mouth and breached shortly after construction following 2 days of high rainfall, and substantial sediment accumulated in the gully above the dam and below the spillway channel on the fan surface; (5) 1987–1993 – the knickpoint retreat continued and the lower fan surface was stable until 1993 when the last signi?cant runoff event overlayed it with c. 10 cm of fresh sediment. These detailed reconstructions of gully development stages allow the contribution of high‐magnitude events to gully growth and regional sediment delivery to be assessed. They further guide management actions to prevent such dam failures in the future. Copyright © 2004 John Wiley & Sons, Ltd.     

Treatment of a Trifluraline Effluent by Means of Oxidation‐Coagulation with Fe(VI) and Combined Fenton Processes

The amination water (AW) effluent stream from the industrial production of the trifluraline herbicide was submitted to an oxidation‐coagulation treatment with potassium ferrate, combined with advanced oxidation processes. The experimental results obtained by analysis of variance (ANOVA) for the oxidation‐coagulation‐Fenton process, evaluating the variables pH (A), Fe(VI) concentration (B), and H₂O₂ concentration (C), demonstrated that the regression equation resulting from the Response Surface Methodology (RSM) experimental design, for the quadratic model, was η_Abs (%) = 36.9– 21.58A + 8.37A² + 1.36B + 0.92B² + 1.08C + 1.52C² + 1.27AB – 1.34AC + 1.33BC. The maximum absorptiometric color reduction occurred at pH 3, with corresponding maximum amounts of iron and hydrogen peroxide. The absorptiometric color and COD reduction were 96% and 57%, respectively. For the oxidation‐coagulation‐photo‐Fenton process, the analyzed variables were pH (A), Fe(VI) concentration (B), H₂O₂ concentration (C), and temperature (D). The regression equation resulting from the quadratic model was η_Abs (%) = 38.3 – 20.2A + 8.12A² – 0.27B + 3.73B² + 0.3C + 3.6C² + 1.67D + 3.1D² + 1.72AB + 0.51AC – 1.82AD + 0.74BC – 1.11BD + 0.03CD. The ANOVA response showed that the highest absorptiometric color reduction occurred at pH 3, with respective maximum amounts of iron and hydrogen peroxide at 60°C. The maximum efficiencies achieved by the proposed treatment process for the trifluraline effluent stream were 95% and 85%, for absorptiometric color and COD reduction, respectively.     

Biodegradation of Petroleum Hydrocarbons by Pseudomonas putida Strain MHF 7109

Pseudomonas putida MHF 7109 has been isolated and identified from cow dung microbial consortium for biodegradation of selected petroleum hydrocarbon compounds – benzene, toluene, and o‐xylene (BTX). Each compound was applied separately at concentrations of 50, 100, 250, and 500 mg L^?1 in minimal salt medium to evaluate degradation activity of the identified microbial strain. The results indicated that the strain used has high potential to degrade BTX at a concentration of 50 mg L^?1 within a period of 48, 96, and 168 h, respectively; whereas the concentration of 100 mg L^?1 of benzene and toluene was found to be completely degraded within 120 and 168 h, respectively. Sixty‐two percent of o‐xylene were degraded within 168 h at the 100 mg L^?1 concentration level. The maximum degradation rates for BTX were 1.35, 1.04, and 0.51 mg L^?1 h^?1, respectively. At higher concentrations (250 and 500 mg L^?1) BTX inhibited the activity of microorganisms. The mass spectrometry analysis identified the intermediates as catechol, 2‐hydroxymuconic semialdehyde, 3‐methylcatechol, cis‐2‐hydroxypenta‐2,4‐dienoate, 2‐methylbenzyl alcohol, and 1,2‐dihydroxy‐6‐methylcyclohexa‐3,5‐dienecarboxylate, for BTX, respectively. P. putida MHF 7109 has been found to have high potential for biodegradation of volatile petroleum hydrocarbons.     

Dissolved Carbohydrate Concentration,Composition, and Bioavailability to Microbial Heterotrophs in Stream Water

Dissolved total carbohydrates (DTCH), dissolved free monosaccharides (DFMS), dissolved organic carbon (DOC), biodegradable DOC (BDOC), and humic substances (HS) were measured in White Clay Creek (WCC), a stream in southeastern Pennsylvania Piedmont, USA. Samples were collected over different seasons and under baseflow and stormflow conditions. DOC concentrations ranged from 1.0 to 12.8 mg/L C with the highest concentrations associated with stormflows. Carbohydrates ranged from 0.42 to 12.4 μM and accounted for 2.9 to 12.7% of the DOC. Humic substances represented the major DOC fraction, accounting for 55 to 72% of the DOC pool under all flow conditions. The humic fraction had a lower carbohydrate content (4.4%) than the non-humic fraction of DOC (7.2%). Stormflow DOC was enriched in carbohydrates relative to baseflow DOC, but the percentage of humic-C changed little. Carbohydrates were primarily present as dissolved polysaccharides (55%), but a significant fraction was bound to humic substances (40%), while a small proportion was present as monosaccharides (5%). The major monosaccharides in stream water, listed in order of decreasing concentration, included glucose, galactose, rhamnose, xylose, arabinose, mannose, and fucose. On average (30.6 ± 7.4)% (n = 44) of the stream water DOC was biodegradable, and carbohydrates accounted for 9.9 to 17.7% of the BDOC.