A novel metabolic pathway for biodegradation of DDT by the white rot fungi, <Emphasis Type="Italic">Phlebia lindtneri</Emphasis> and <Emphasis Type="Italic">Phlebia brevispora</Emphasis>

1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) was used as the substrate for a degradation experiment with the white rot fungi Phlebia lindtneri GB-1027 and Phlebia brevispora TMIC34596, which are capable of degrading polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated biphenyls (PCBs). Pure culture of P. lindtneri and P. brevispora with DDT (25 μmol l⁻¹) showed that 70 and 30% of DDT, respectively, disappeared in a low-nitrogen medium after a 21-day incubation period. The metabolites were analyzed using gas chromatography/mass spectrometry (GC/MS). Both fungi metabolized DDT to 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 2,2-bis(4-chlorophenyl)acetic acid (DDA) and 4,4-dichlorobenzophenone (DBP). Additionally, DDD was converted to DDA and DBP. DDA was converted to DBP and 4,4-dichlorobenzhydrol (DBH). While DBP was treated as substrate, DBH and three hydroxylated metabolites, including one dihydroxylated DBP and two different isomers of monohydroxylated DBH, were produced from fungal cultures, and these hydroxylated metabolites were efficiently inhibited by the addition of a cytochrome P-450 inhibitor, piperonyl butoxide. These results indicate that the white rot fungi P. lindtneri and P. brevispora can degrade DBP/DBH through hydroxylation of the aromatic ring. Moreover, the single-ring aromatic metabolites, such as 4-chlorobenzaldehyde, 4-chlorobenzyl alcohol and 4-chlorobenzoic acid, were found as metabolic products of all substrate, demonstrating that the cleavage reaction of the aliphatic-aryl carbon bond occurs in the biodegradation process of DDT by white rot fungi.     

Isolation and characterization of a fungus <Emphasis Type="Italic">Aspergillus</Emphasis> sp. strain F-3 capable of degrading alkali lignin

A fungus strain F-3 was selected from fungal strains isolated from forest soil in Dalian of China. It was identified as one Aspergillus sp. stain F-3 with its morphologic, cultural characteristics and high homology to the genus of rDNA sequence. The budges or thickened node-like structures are peculiar structures of hyphae of the strain. The fungus degraded 65% of alkali lignin (2,000 mg l⁻¹) after day 8 of incubation at 30°C at pH 7. The removal of colority was up to 100% at 8 days. The biodegradation of lignin by Aspergillus sp. F-3 favored initial pH 7.0. Excess acid or alkali conditions were not propitious to lignin decomposing. Addition of ammonium l-tartrate or glucose delayed or repressed biodegradation activities. During lignin degradation, manganese peroxidase (28.2 U l⁻¹) and laccase (3.5 U l⁻¹)activities were detected after day 7 of incubation. GC-MS analysis of biodegraded products showed strain F-3 could convert alkali lignin into small molecules or other utilizable products. Strain F-3 may co-culture with white rot fungus and decompose alkali lignin effectively.     

Decolorizing Reactive Textile Dyes with White‐Rot Fungi by Temporary Immersion Cultivation

The decontamination of effluents from textile industries is problematic due to the fact that textile dyes are resistant to degradation in the environment. Enzymes from white rot fungi, especially laccase, are able to degrade various complex aromatic structures, and are therefore able to decolorize textile dyes. The white‐rot fungi Trametes versicolor and Phanerochaete chrysosporium were immobilized, separately, on both pine wood chips and palm oil fiber, and cultivated in the temporary immersion RITA® (Récipient à Immersion Temporaire Automatique) System, which was adapted to serve as a fungal bioreactor in a series of four experiments to determine optimal conditions for decolorizing the textile dyes Levafix Blue and Remazol Brilliant Red. The maximum rate of decolorization of both dyes occurred within 24 h of incubation, and laccase was detected in the system.     

Textile dye decolorization using cyanobacteria

Cyanobacterial cultures isolated from sites polluted by industrial textile effluents were screened for their ability to decolorize cyclic azo dyes. Gloeocapsa pleurocapsoides and Phormidium ceylanicum decolorized Acid Red 97 and FF Sky Blue dyes by more than 80% after 26 days. Chroococcus minutus was the only culture which decolorized Amido Black 10B (55%). Chlorophyll a synthesis in all cultures was strongly inhibited by the dyes. Visible spectroscopy and TLC confirmed that color removal was due to degradation of the dyes.Revisions requested 10 November 2004/30 November 2004; Revisions received 16 November 2004/ 7 January 2005     

Gravimetric screening method for fungal decay of paper: inoculation with <Emphasis Type="Italic">Trametes</Emphasis> <Emphasis Type="Italic">versicolor</Emphasis>

The European standard test EN 113 for fungal degradation of solid wood has been adapted for degradation of paper by white rot fungus (Trametes versicolor). Fungal degradation of paper sheets may potentially be used for screening different wood preservatives on paper instead of solid wood. The paper samples showed higher relative mass losses compared to wood, and samples pretreated with boric acid, copper sulfate and polymerized linseed oil were successfully tested for biodegradation using the paper sheet method. The results on paper degradation were compared with wood, both as wood blocks (according to standard test) and wood cut in sections forming layered structures mimicking paper layers.     

Degradation of azo dyes containing aminonaphthol by Sphingomonas sp strain 1CX

Sphingomonas sp strain 1CX was isolated from a wastewater treatment plant and is capable of aerobically degrading a suite of azo dyes, using them as a sole source of carbon and nitrogen. All azo dyes known to be decolorized by strain 1CX (Orange II, Acid Orange 8, Acid Orange 10, Acid Red 4, and Acid Red 88) have in their structure either 1-amino-2-naphthol or 2-amino-1-naphthol. In addition, an analysis of the structures of the dyes degraded suggests that there are certain positions and types of substituents on the azo dye which determine if degradation will occur. Growth and dye decolorization occurs only aerobically and does not occur under fermentative or denitrification conditions. The mechanism by which 1CX decolorizes azo dyes appears to be through reductive cleavage of the azo bond. In the case of Orange II, the initial degradation products were sulfanilic acid and 1-amino-2-naphthol. Sulfanilic acid, however, was not used by 1CX as a growth substrate. The addition of glucose or inorganic nitrogen inhibited growth and decoloration of azo dyes by 1CX. Attempts to grow the organism on chemically defined media containing several different amino acids and sugars as sources of nitrogen and carbon were not successful. Phylogenetic analysis of Sphingomonas sp strain 1CX shows it to be related to, but distinct from, other azo dye-decolorizing Sphingomonas spp strains isolated previously from the same wastewater treatment facility. Received 19 May 1999/ Accepted in revised form 11 August 1999     

Biodegradation of γ-hexachlorocyclohexane (Lindane) by a non-white rot fungus conidiobolus 03-1-56 isolated from litter

Biodegradation of chlorinated pesticide γ-hexachlorocyclohexane (lindane) by a nonwhite rot fungus Conidiobolus 03-1-56 is reported for the first time. Conidiobolus 03-1-56, a phycomyceteous fungus isolated from litter, completely degraded lindane on the 5th day of incubation in the culture medium, and GC-ECD studies confirmed that lindane removal did not occur via adsorption on the fungal biomass. Degradation studies using different medium compositions showed that nitrogen/carbon limiting conditions (stress conditions) and presence of veratryl alcohol, induced the secretion of extracellular oxidative enzymes, which enhanced the rate of lindance biodegradation. Under optimum nutrient-limiting conditions, GC-ECD and GC-MS analysis showed complete absence of any degradation metabolite, indicating that lindane was completely mineralized. Assays for tannic acid utilization and lignin peroxidase showed similar enzymatic profiles between Conidiobolus 03-1-56 and standard white rot fungi Pleurotus ostreatus 1200 and Trametes versicolor 1086. Although Conidiobolus 03-1-56 showed a reduced enzyme activity compared to white rot fungi, preliminary evidence indicates that enzymes responsible for lignin degradation by white rots play a key role in lindane degradation by Conidiobolus 03-1-56.     

Aerobic biodegradation of a mixture of sulfonated azo dyes by a bacterial consortium immobilized in a two‐stage sparged packed‐bed biofilm reactor

The biodegradation of the sulfonated azo dyes, Acid Orange 7 (AO7) and Acid Red 88 (AR88), by a bacterial consortium isolated from water and soil samples obtained from sites receiving discharges from textile industries, was evaluated. For a better removal of azo dyes and their biodegradation byproducts, an aerobically operated two‐stage rectangular packed‐bed biofilm reactor (2S‐RPBR) was constructed. Because the consortium's metabolic activity is affected by oxygen, the effect of the interstitial air flow rate Q_GI on 2S‐RPBR's zonal values of the oxygen mass transfer coefficient k_La was estimated. In the operational conditions probed in the bioreactor, the k_La values varied from 3 to 60 h^?1, which roughly correspond to volumetric oxygen transfer rates, dc_L/dt, ranging from 20 to 375 mg O₂ L^?1h^?1. Complete biodegradation of azo dyes was attained at loading rates B_V,AZ up to 40 mg L^?1d^?1. At higher B_V,AZ values (80 mg L^?1 d^?1), dye decolorization and biodegradation of the intermediaries 4‐amino‐naphthalenesulphonic acid (4‐ANS) and 1‐amino‐2‐naphthol (1‐A2N) was almost complete. However, a diminution in COD and TOC removal efficiencies was observed in correspondence to the 4‐aminobenzenesulfonic acid (4‐ABS) accumulation in the bioreactor. Although the oxygen transport rate improved the azo dye mineralization, the results suggest that the removal efficiency of azo dyes was affected by biofilm detachment at relatively high Q_GI and B_V,AZ values. After 225 days of continuous operation of the 2S‐RFBR, eight bacterial strains were isolated from the biofilm attached to the porous support. The identified genera were: Arthrobacter, Variovorax, Agrococcus, Sphingomonas, Sphingopyxis, Methylobacterium, Mesorhizobium, and Microbacterium.     

Detoxification and mineralization of Acid Blue 74: study of an alternative secondary treatment to improve the enzymatic decolourization

Many reports describe the decolourization of dyes by fungal enzymes. However, these enzymes do not contribute to dye mineralization but only to its biotransformation into less coloured or colourless molecules persisting in solution. Therefore, it is essential to analyse the identity of the metabolites produced during enzymatic treatments and its biodegradation into an appropriate system. The present work examines the decolourization/detoxification of a simulated effluent (containing Acid Blue 74) by fungal enzymes and proposes a secondary treatment using an anaerobic system to improve the enzymatic decolourization through the complete mineralization of the dye. Ligninolytic enzymes were produced by solid culture using the thermo-tolerant fungus Fomes sp. EUM1. The enzymes produced showed a high rate of decolourization (>95 % in 5 h) and were stable at elevated temperature (40 °C) and ionic strength (NaCl, 50 mM). Isatin-5-sulphonic acid was identified via ¹H-NMR as oxidation product; tests using Daphnia magna revealed the non-toxic nature of this compound. To improve the enzymatic degradation and avoid coupling reactions between the oxidation products, the effluent was subjected to an anaerobic (methanogenic) treatment, which achieved high mineralization efficiencies (>85 %). To confirm the mineralization of isatin-5-sulphonic acid, a specific degradation study, which has not been reported before, with this single compound was conducted under the same conditions; the results showed high removal efficiencies (86 %) with methane production as evidence of mineralization. These results showed the applicability of an anaerobic methanogenic system to improve the enzymatic decolourization/detoxification of Acid Blue 74 and achieve its complete mineralization.     

Synthetic dye decolourization by white rot fungi

Synthetic dyes are integral part of many industrial products. The effluents generated from textile dyeing units create major environmental problems and issues both in public and textile units. Industrial wastewater treatment is one of the major problems in the present scenario. Though, the physical and chemical methods offer some solutions to the problems, it is not affordable by the unit operators. Biological degradation is recognized as the most effective method for degrading the dye present in the waste. Research over a period of two decades had provided insight into the various aspects of biological degradation of dyes. It is observed that the white rot fungi have a non-specific enzyme system, which oxidizes the recalcitrant dyes. Detailed and extensive studies have been made and process developed for treatment of dye containing wastewaters by white rot fungi and their enzyme systems. An attempt is made to summarize the detailed research contributions on these lines.     

Potential of aquatic fungi derived from diverse freshwater environments to decolourise synthetic azo and anthraquinone dyes

A total of 37 strains of aquatic hyphomycetes and 95 fungal isolates derived from diverse freshwater environments were screened on agar plates for the decolourisation of the disazo dye Reactive Black 5 and the anthraquinone dye Reactive Blue 19. The decolourisation of 9 azo and 3 anthraquinone dyes by 9 selected aquatic fungi was subsequently assessed in a liquid test system. The fungi were representatives of mitosporic anamorphs, and 6 strains had proven ascomycete affiliations. For comparison, 5 white rot basidiomycetes were included. The majority of dyes were decolourised by several mitosporic aquatic isolates at rates essentially comparable to those observed with the most efficient white rot fungus. Under certain conditions, particular aquatic strains decolourised dyes even more efficiently than the best performing white rot basidiomycete. Upon fungal treatment of several dyes, new absorbance peaks appeared, indicating biotransformation metabolites. All together, these results point to the potential of fungi occurring in freshwater environments for the treatment of dye-containing effluents.     

Fungal pretreatment of lignocellulosic biomass

Pretreatment is a crucial step in the conversion of lignocellulosic biomass to fermentable sugars and biofuels. Compared to thermal/chemical pretreatment, fungal pretreatment reduces the recalcitrance of lignocellulosic biomass by lignin-degrading microorganisms and thus potentially provides an environmentally-friendly and energy-efficient pretreatment technology for biofuel production. This paper provides an overview of the current state of fungal pretreatment by white rot fungi for biofuel production. The specific topics discussed are: 1) enzymes involved in biodegradation during the fungal pretreatment; 2) operating parameters governing performance of the fungal pretreatment; 3) the effect of fungal pretreatment on enzymatic hydrolysis and ethanol production; 4) efforts for improving enzymatic hydrolysis and ethanol production through combinations of fungal pretreatment and physical/chemical pretreatment; 5) the treatment of lignocellulosic biomass with lignin-degrading enzymes isolated from fungal pretreatment, with a comparison to fungal pretreatment; 6) modeling, reactor design, and scale-up of solid state fungal pretreatment; and 7) the limitations and future perspective of this technology.     

Biodegradation of textile azo dye by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12 under microaerophilic conditions

The complete biodegradation of azo dye, Fast Acid Red GR, was observed under microaerophilic conditions by Shewanella decolorationis S12. Although the highest decolorizing rate was measured under anaerobic condition and the highest biomass was obtained under aerobic condition, a further biodegradation of decolorizing products can only be achieved under microaerophilic conditions. Under microaerophilic conditions, S. decolorationis S12 could use a range of carbon sources for azo dye decolorization, including lactate, formate, glucose and sucrose, with lactate being the optimal carbon source. Sulfonated aromatic amines were not detected during the biotransformation of Fast Acid Red GR, while H₂S formed. The decolorizing products, aniline, 1,4-diaminobenzene and 1-amino-2-naphthol, were followed by complete biodegradation through catechol and 4-aminobenzoic acid based on the analysis results of GC-MS and HPLC.     

Degradation of ferulic acid by a white rot fungus <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

Summary A ubiquitous white rot fungus Schizophyllum commune was used for the first time to study the degradation of ferulic acid. Vanillic acid was observed as one of the major products of ferulic acid catabolism, with vanillin formed as an intermediate. Almost 99.9% ferulic acid with a initial concentration of 5 mM was consumed by this fungus after 16 days of incubation at 37 °C.     

Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions

The brown rot fungus Wolfiporia cocos and the selective white rot fungus Perenniporia medulla-panis produce peptides and phenolate-derivative compounds as low molecular weight Fe³⁺-reductants. Phenolates were the major compounds with Fe³⁺-reducing activity in both fungi and displayed Fe³⁺-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe³⁺ and H₂O₂ (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum—a model brown rot fungus—other brown rot fungi as well as selective white rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in brown rot fungi, and lignin-modifying enzymes in white rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to protect wood from degradation by microorganisms.     

Isolation and selection of novel basidiomycetes for decolorization of recalcitrant dyes

Thirty wood-rotting basidiomycetes, most of them causing white rot in wood, were isolated from fruiting bodies growing on decaying wood from the Sierra de Ayllón (Spain). The fungi were identified on the basis of their morphological characteristics and compared for their ability to decolorize Reactive Black 5 and Reactive Blue 38 (as model of azo and phthalocyanine type dyes, respectively) at 75 and 150 mg/L. Only eighteen fungal strains were able to grow on agar plates in the presence of the dyes and only three species (Calocera cornea, Lopharia spadicea, Polyporus alveolaris) decolorized efficiently both dyes at both concentrations. The ligninolytic activities, involved in decolorization dyes (laccases, lignin peroxidases, Mn-oxidizing peroxidases), were followed in glucose basal medium in the presence of enzyme inducers. The results indicate a high variability of the ligninolytic system within white-rot basidiomycetes. These fungal species and their enzymes can represent new alternatives for the study of new biological systems to degrade aromatic compounds causing environmental problems.     

Decolorization of Several Polymeric Dyes by the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

The polymeric dyes Poly B-411, Poly R-481, and Poly Y-606 were examined as possible alternatives to the radiolabeled lignin previously used as a substrate in lignin biodegradation assays. Like lignin degradation, the decolorization of these dyes by the white rot basidiomycete Phanerochaete chrysosporium occurred during secondary metabolism, was suppressed in cultures grown in the presence of high levels of nitrogen, and was strongly dependent on the oxygen concentration in the cultures. A variety of inhibitors of lignin degradation, including thiourea, azide, and 4′-O-methylisoeugenol, also inhibited dye decolorization. A pleiotropic mutant of P. chrysosporium, 104-2, lacking phenol oxidase and ligninolytic activity was also not able to decolorize the polymeric dyes, whereas a phenotypic revertant strain, 424-2, regained this capacity. All of these results suggest that the ligninolytic degradation activity of the fungus was responsible for the decolorization of these dyes.     

Biodegradation of tetracycline by the yeast strain Trichosporon mycotoxinivorans XPY-10

We investigated the behavior of tetracycline degradation and its degradation products upon treatment of isolated yeast that we termed “XPY-10.” XPY-10 was isolated from wastewater and identified as Trichosporon mycotoxinivorans by morphological and physiological tests and 5.8S rRNA ITS sequencing. In our experiments, 78.28 ± 0.8% of tetracycline was removed within 7 days with XPY-10. The degradation of tetracycline fitted well with the first-order kinetic model. We also speculated upon the biodegradation products formed during biodegradation. The possible structures of five products were determined using liquid chromatography–tandem mass spectrometry. During practical application, XPY-10 was shown to have an obvious influence on biodegradation, and 89.61% of tetracycline was removed in feedlot sewage after 7 days of reaction. The chemical oxygen demand removal reached 73.47%.     

Degradation of ochratoxin a and b by the white rot fungus<Emphasis Type="Italic">pleurotus ostreatus</Emphasis>

Degradation of ochratoxin A (OTA) and B (OTB) by three selected fungi during solid state fermentation of barley contaminated with ochratoxins was compared. In presence of the soil fungusRhizopus japonicus and the white rot fungusPanerochaete chrysosporium more than 60 % of the mycotoxins remained stable, while in the white rot fungusPleurotus ostreatus only 23 % of the initial OTA and 3 % of OTB were detected after a four weeks incubation period. Kinetic studies on mycotoxin degradation byPI ostreatus demonstrated formation of ochratoxin α and presumably ochratoxin β as intermediate products, what indicates that hydrolysis is the first step in OTA and OTB degradation followed by further degradation of the intermediates.     

Laccase production and simultaneous decolorization of synthetic dyes in unique inexpensive medium by new isolates of white rot fungus

Morphological and biochemical analysis of the newly isolated white rot fungal (WRF-1) strain has ability to secrete laccase in the economical medium consisted of synthetic dyes, groundnut shell (GNS) and cyanobacterial biomass (algal bloom) under submerged shaking condition at pH 5.0 and 30 °C ± 2 °C temperature. WRF-1 strain was found to decolorize synthetic dyes efficiently at pH 5.0 and 30 °C ± 2 °C temperature. The laccase activity of strain was purified to homogeneity by chromatography with yield up to 70%. The molecular mass of laccase was found to be 70 kDa by SDS-PAGE and isoelectric point was 4.8. Biotransformation of the dyes was followed spectrophotometrically and dyes were found to decolorize completely after 6 days of fermentation. LC-MS studies were used to decipher the degradation profile of synthetic dyes by WRF-1. Indigo carmine gets degraded to isatin sulfonic acid and 4-amino-3-methylbenzenesulphonic acid whereas methyl orange degraded metabolites were identified as p-N,N′-dimethylamine phenyldiazine and p-hydroxybenzene sulfonic acid. Thus the study would give a road map for the production and application of laccase enzyme on a larger scale using low cost substrate.