Influence of Agitation on the Removal of Nonylphenol by the White-rot Fungi Trametes versicolor and Bjerkandera sp. BOL 13

Bjerkandera sp. BOL 13 removed 95% of nonylphenol (at 9.7 mg nonylphenol l⁻¹ day⁻¹) from aqueous medium after 5 days of incubation in agitated cultures. This removal rate decreased 2.5-fold in static cultures. By comparison, Trametes versicolor removed nonylphenol at 2.8 mg l⁻¹ day⁻¹ under conditions of static incubation, probably due to the action of laccase, but no growth was recorded in the agitated bottles. Received 4 August 2005; Revisions requested 18 August 2005 and 27 September 2005; Revisions received 22 September 2005 and 3 November 2005; Accepted 7 November 2005     

Isolation and characterization of a white rot fungus <Emphasis Type="Italic">Bjerkandera</Emphasis> sp. strain capable of oxidizing phenanthrene

Strain BOL13 was selected from 18 fungal strains isolated from an oil-spill contaminated site in Oruro, Bolivia. It was identified as a basidiomycete with high homology to Bjerkandera. The fungus degraded 100 mg phenanthrene l⁻¹ at 0.17 mg l⁻¹ d⁻¹ at 30 °C at pH 7. During phenanthrene degradation, a maximum manganese peroxidase activity of 100–120 U l⁻¹ was measured after 10 days of incubation. The ability of Bjerkandera sp. to produce lignin-modifying enzymes and to oxidize phenanthrene under various pH and temperature conditions was confirmed.     

Enhanced production of laccase activity by <Emphasis Type="Italic">Trametes versicolor </Emphasis>immobilized into alginate beads by the addition of different inducers

In the present paper the effect of adding veratryl alcohol and copper sulphate on laccase activity production by Trametes versicolor immobilized into alginate beads has been investigated. Employing copper sulphate as laccase inducer or supplementing the culture medium with veratryl alcohol, led to maximum values of laccase activity. However, the highest laccase activity (around 4,000 U l⁻¹) was obtained in cultures simultaneously supplemented with copper sulphate (3 mM) and veratryl alcohol (20 mM). These values implied a considerable enhancement in relation to␣control cultures without any inducer (around 200 U l⁻¹). The production of laccase by immobilized T. versicolor in a 2-l airlift bioreactor with the optimized inducer has been evaluated. Laccase activities around 1,500 U l⁻¹ were attained. The bioreactor operated for 44 days without operational problems and the bioparticles (fungus grows in alginate beads) maintained their shape throughout the fermentation. Moreover, the extracellular liquid obtained was studied in terms of pH and temperature activity and stability. On the other hand, anthracene was added in two-repeated batches in order to determine the efficiency of this process to degrade pollutants. Near complete degradation was reached in both batches. Moreover, in vitro degradation of several polycyclic aromatic hydrocarbons by crude laccase was also performed.     

Biodegradation of nonylphenol in a continuous packed-bed bioreactor

A packed bed bioreactor, with 170 ml glass bead carriers and 130 ml medium, was tested for the removal of the endocrine disrupter, nonylphenol, with a Sphingomonas sp. The bioreactor was first continuously fed with medium saturated with nonylphenol in an attempt to simulate groundwater pollution. At best, nonylphenol was degraded by 99.5% at a feeding rate of 69 ml h^–1 and a removal rate of 4.3 mg nonylphenol day^–1, resulting in a 7.5-fold decrease in effluent toxicity according to the Microtox. The bioreactor was then fed with soil leachates at 69 ml h^–1 from artificially contaminated soil (1 g nonylphenol kg^–1 soil) and a real contaminated soil (0.19 g nonylphenol kg^–1 soil). Nonylphenol was always completely removed from the leachates of the two soils. It was removed by 99% from the artificial soil but only 62% from real contaminated soil after 18 and 20 d of treatment, respectively, showing limitation due to nonylphenol adsorption.     

Continuous decolorization of bleached kraft effluents byCoriolus versicolor in the form of pellets

Summary Continuous decolorization of kraft black liquor by mycelial pellets ofCoriolus versicolor in the presence of glucose as co-substrate is discussed. A linear relationship was developed between the rate of decolorization and the liquor concentration. The rate constant was equal to 0.00961 gmyc^–1 h^–` at 22°C. During the continuous experiments the pellets exhibited no apparent loss of activity when the liquor concentration was in the range of 400 CU l^–1 to 5000 CU l^–1. However, in the repeated batch experiments a loss of activity was observed which was dependent on the initial liquor concentration. The half-life of pellets was equal to 4.7, 9.4 and 20.2 days for the initial liquor concentration of 1380, 31 780 and 6990 CU l^–1, respectively. The production of the extracellular enzyme, laccase, was followed but could not be used as an indicator of the ligninolytic activity. The involvement of the intracellular enzymes ofC. versicolor in the decolorization process is described.     

Aerobic biodegradation of nonylphenol by cold-adapted bacteria

Three strains capable of mineralizing nonylphenol as sole carbon source were isolated from a sample of contaminated soil and characterized as two Pseudomonas spp. and a Stenotrophomonas sp. The two Pseudomonas spp. expressed characteristics typical of psychrophiles growing optimally of 10 °C and capable of growing at 0 °C. The Stenotrophomonas sp. was more likely psychrotrophic because it had an optimal temperature between 14 and 22 °C although it was not capable of growing at 4 °C. At 14 °C, one of the Pseudomonas spp. exhibited the highest rate of degradation of nonylphenol (4.4 mg l^–1 d^–1), when compared with axenic or mixed cultures of the isolates. This study represents, to the best of our knowledge, the first reported case of cold-adapted microorganisms capable of mineralizing nonylphenol.     

Selective control of cyanobacteria by surfactin-containing culture broth of Bacillus subtilis C1

Of several types of chemical surfactants and biosurfactants, only the culture broth of Bacillus subtilis C1 containing surfactin at 10 mg l^–1 completely inhibited the growth of Microcystis aeruginosa, a bloom-forming cyanobacterium in highly eutrophic lakes. The broth with 10 mg surfactin l^–1 also removed 85% of the maximally grown M. aeruginosa (chlorophyll-a concentration, 1000 g l^–1) within 2 d, and the removal efficiency was enhanced by Ca²⁺ over 1 mM. The growth of Anabaena affinis, another bloom-forming cyanobacterium, was also inhibited about 70% with surfactin at 10 mg l^–1 broth. However, the effect of the broth was delayed over 3 d in the green algae, Chlorella vulgaris and Scenedesmus sp., and was negligible in a diatom, Navicula sp., indicating the potential for the selective control of cyanobacterial blooms.     

Potential for bioremediation of agro-industrial effluents with high loads of pesticides by selected fungi

Wastewaters from the fruit packaging industry contain a high pesticide load and require treatment before their environmental discharge. We provide first evidence for the potential bioremediation of these wastewaters. Three white rot fungi (WRF) (Phanerochaete chrysosporium, Trametes versicolor, Pleurotus ostreatus) and an Aspergillus niger strain were tested in straw extract medium (StEM) and soil extract medium (SEM) for degrading the pesticides thiabendazole (TBZ), imazalil (IMZ), thiophanate methyl (TM), ortho-phenylphenol (OPP), diphenylamine (DPA) and chlorpyrifos (CHL). Peroxidase (LiP, MnP) and laccase (Lac) activity was also determined to investigate their involvement in pesticide degradation. T. versicolor and P. ostreatus were the most efficient degraders and degraded all pesticides (10 mg l⁻¹) except TBZ, with maximum efficiency in StEM. The phenolic pesticides OPP and DPA were rapidly degraded by these two fungi with a concurrent increase in MnP and Lac activity. In contrast, these enzymes were not associated with the degradation of CHL, IMZ and TM implying the involvement of other enzymes. T. versicolor degraded spillage-level pesticide concentrations (50 mg l⁻¹) either fully (DPA, OPP) or partially (TBZ, IMZ). The fungus was also able to rapidly degrade a mixture of TM/DPA (50 mg l⁻¹), whereas it failed to degrade IMZ and TBZ when supplied in a mixture with OPP. Overall, T. versicolor and P. ostreatus showed great potential for the bioremediation of wastewaters from the fruit packaging industry. However, degradation of TBZ should be also achieved before further scaling up.     

Decolorization of phenolic effluents by soluble and immobilized phenol oxidases

Summary Colour removal from phenplic industrial effluents by phenol oxidase enzymes and white-rot fungi was compared. Soluble laccase and horseradish peroxidase (HRP) removed colour from pulp mill (E), cotton mill hydroxide (OH) and cotton mill sulphide (S) effluents, but rapid and irreversible enzyme inactivation took place. Entrapment of laccase in alginate beads improved decolorization by factors of 3.5 (OH) and 2 (E); entrapment of HRP improved decolorization by 36 (OH), 20 (E) and 9 (S). Beads were unsuitable for continuous use because the enzymes were rapidly released into solution. Co-polymerization of laccase or HRP with L-tyrosine gave insoluble polymers with enzyme activity. Entrapment of the co-polymers in gel beads further increased the efficiency of decolorization of E by 28 (laccase) and by 132 (HRP) compared with soluble enzymes. Maximum decolorization of all three effluents by batch cultures of Coriolus versicolor (70%–80% in 8 days) was greater than the maximum enzymic decolorization (48% of OH in 3 days by entrapped laccase). Soluble laccase (222 units ml^–1) precipitated 1.2 g l^–1 phenol from artificial coal conversion effluent at pH 6.0 and the rate of precipitation and enzyme inactivation was faster at pH 6.0 than at pH 8.5.Offprint requests to: R. G. Burns     

Degradation of sodium anthraquinone sulphonate by free and immobilized bacterial cultures

Aerobic biodegradation of a xenobiotic recalcitrant compound sodium anthraquinone-2-sulphonate (SAS), was investigated using as an inoculum a mixed microbial culture, which was activated sludge from industrial and domestic waste-water treatment plants. The difference in SAS degradation was examined using two main systems: (1) suspended cells and (2) immobilized cells, both in batch and in continuous culture. In the suspended cell system, under continuous culture conditions using SAS as a unique source of carbon and energy, it was possible to degrade about 95% of this substrate after 6 days. Maximal SAS removal rates in the suspended-cell system were 593 mg SAS l^–1 h^–1 and 88.7 mg SAS l^–1 h^–1 for dilution rates (D) of 0.05 h^–1 and 0.075 h^–1, respectively. In the immobilized-cell system, almost all SAS was degraded in 6 days and the maximal removal rate reached 88.7 mg SAS l^–1 h^–1 at D=0.05 h^–1. Application of a continuous-flow enrichment procedure resulted in selection of several kinds of micro-organisms and led to a progressive elimination of some species of Aeromonas. A stable microbial community of 11 strains has been established and characterized at D=0.075 h^–1. Most of them were Gram-negative and belonged to the genus Pseudomonas.     

Enrichment and kinetics of biodegradation of 1-naphthylamine in activated sludge

Sequencing-batch reactors were used to develop an activated sludge enrichment culture capable of degrading 1-naphthylamine (1NA). Approximately 5 months acclimation with salicylic acid (1600 mg l^–1) as the primary source of carbon were required to obtain an enrichment culture able to degrade even small quantities of 1NA. After an additional 4 months acclimation, during which the concentration of salicyclic acid was decreased to 50 mg l^–1, a culture developed that degraded 1NA concentrations as high as 300 mg l^–1. Kinetic determinations showed that 1NA degradation (in the presence of salicylate) followed Michaelis-Menten kinetics with K _m and V _m values of 32.5±2.2 mg l^–1 and 375±18 ng 1NA mg^–1 cells h^–1, respectively. The same enrichement was able to degrade 1NA when present as the sole source of carbon and energy and to convert approximately 87% to CO₂.     

Overproduction of recombinant laccase using a homologous expression system in <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants g^–1 of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.     

Bioconversion of compactin into pravastatin by Streptomyces sp.

Streptomyces sp. Y-110, isolated from soil, modified compactin to pravastatin, a therapeutic agent for hypercholesterolemia. In a batch culture, the highest production of pravastatin was 340 mg l^–1 from 750 mg compactin l^–1 in 24 h. By intermittent feeding of compactin into the culture medium, both the compactin concentration and its conversion increased to 2000 mg l^–1 and 1000 mg pravastatin l^–1, respectively, with the conversion rate of 10 mg l^–1 h^–1. Continuous feeding of compactin increased production of pravastatin to 15 mg l^–1 h^–1.     

Degradative ability of 2,4,6-tribromophenol by saprophytic fungi Trametes versicolor and Agaricus augustus isolated from chilean forestry

Trametes versicolor and Agaricus augustus, with a maximum tolerable concentration (MTC) of 80 μg ml⁻¹ tribromophenol (TBP), were selected to evaluate TBP biodegradation capacity. These fungi were capable of decreased TBP concentrations and A. augustus was also capable of biotransforming TBP to tribromoanisole (TBA). Peroxidase and laccase activities were observed in the T. versicolor supernatant but not in that of A. augustus. These tolerance levels could be due to either lignolytic enzymes that degrade TBP or the ability of the fungi to biotransform TBP to tribromoanisole, respectively. The sustained ability of T. versicolor to degrade TBP (total of 40 μg ml⁻¹) in the presence of an additional carbon source suggests that it may have potential applications in the degradation of forestry industry waste.     

Isolation and characterization of sulfur-utilizing denitrifiers from the sulfur-oxidizing denitrification process

Of 14 potential sulfur-oxidizing strains, Pseudomonas sp. B21 and Agrobacterium sp. B19 were considered as denitrifiers. Under aerobic conditions, with S⁰ as electron donor, maximum cell growth rates were 0.022 (B21) and 0.043 h^–1 (B19). Both grew optimally at pH 7.5 and 28 °C. When NO₃-N was increased from 10 to 200 mg l^–1 the efficiency of nitrate removal of each strain gradually decreased, from 60 to 40%. Addition of suitable organic compounds (C/N < 4.2) increased the nitrate removal efficiencies of both strains, indicating their mixotrophic characters.     

Continuous acetonitrile degradation in a packed-bed bioreactor

A 20-l packed-bed reactor filled with foamed glass beads was tested for the treatment of acetonitrile HPLC wastes. Aeration was provided by recirculating a portion of the reactor liquid phase through an aeration tank, where the dissolved oxygen concentration was kept at 6 mg/l. At a feeding rate of 0.77 g acetonitrile l^–1 reactor day^–1, 99% of the acetonitrile was removed; and 86% of the nitrogen present in acetonitrile was released as NH₃, confirming that acetonitrile volatilization was not significant. Increasing the acetonitrile loading resulted in lower removal efficiencies, but a maximum removal capacity of 1.0 g acetonitrile l^–1 reactor day^–1 was achieved at a feeding rate of 1.6 g acetonitrile l^–1 reactor day^–1. The removal capacity of the system was well correlated with the oxygenation capacity, showing that acetonitrile removal was likely to be limited by oxygen supply. Microbial characterization of the biofilm resulted in the isolation of a Comamonas sp. able to mineralize acetonitrile as sole carbon, nitrogen and energy source. This organism was closely related to C. testosteroni (91.2%) and might represent a new species in the Comamonas genus. This study confirms the potential of packed-bed reactors for the treatment of a concentrated mixture of volatile pollutants.     

Degradation of carbondisulphide by a Thiobacillus isolate

During experiments investigating the purification of waste gas a bacterium capable of using carbon disulphide (CS₂) als sole energy source was isolated. It could be identified as a Thiobacillus sp.; however, the species remains unclear. Both the properties of T. thioparus and T. thiooxidans have been observed. Since the organism could be used for removing CS₂ in the environment, the degradation kinetics have been investigated by different methods. Substrate concentrations of up to 100 mg CS₂·l^–1 were oxidized at maximum rates of 2.5 mg CS₂·g^–1 protein·min^–1 at pH 7.0 and at 30°C. CS₂ levels above 150 mg CS₂·l^–1 caused termination of degradative activity. Correspondence to: Ch. Plas     

Removal of inorganic nitrogen sources from water by the algal biofilm of the aerial microalga Trentepohlia aurea

Removal of inorganic nitrogen sources by cells of the aerial microalga Trentepohlia aurea grown on the surface of substrate, such as filter paper, has been investigated in a batch system. When the alga grew on the paper dampened with medium, it actively ingested inorganic nitrogenous compounds in the medium. Immobilized cells on the filter papers were called algal biofilm in this study. When the algal biofilms were soaked in modified Bold's Basal medium (using 1 g NH₄Cl l^–1 as a N source), the removal rate was 4.25 mg ammonium-N l^–1 day^–1 in 40 days. In modified medium with added 26 mg nitrite-N, the removal rate of the total inorganic N ion by the biofilms reached 5.11 mg N l^–1 day^–1. This removal rate of total N ion was higher than that in the medium by addition of 26 mg nitrate-N. In addition, we tried to examine simultaneous removal of ammonium, nitrate, and nitrite ions and growth inhibition of cyanobacteria in the medium by using the algal biofilms. Consequently, it was demonstrated that the algal biofilms of T. aurea could be utilized as a biofunctional material for the purification of wastewater.     

Evaluation of different fungal strains in the decolourisation of synthetic dyes

Of seven fungal strains tested for their ability to decolourise three structurally diverse synthetic dyes, Phanerochaete sordida, Bjerkandera sp. BOS55, Phlebia radiata, and Phanerochaete chrysosporium had average values of maximum decolourisation rates higher than 0.2 [Absorbance] d^–1. All seven fungi produced manganese peroxidase (MnP) but laccase activity was detected only in Phlebia radiata. No lignin peroxidase (LiP) activity was observed.     

Optimization of manganese peroxidase and laccase production in the South American fungus<Emphasis Type="Italic"> Fomes sclerodermeus</Emphasis> (Lév.) Cke

Fomes sclerodermeus produces manganese peroxidase (MnP) and laccase as part of its ligninolytic system. A Doehlert experimental design was applied in order to find the optimum conditions for MnP and laccase production. The factors studied were Cu²⁺, Mn²⁺ and asparagine. The present model and data analysis allowed us not only to define optimal media for production of both laccase and MnP, but also to show the combined effects between the factors. MnP was strongly influenced by Mn²⁺, which acts as an inducer. Under these conditions Cu²⁺ negatively affected MnP activity. At 13 days of growth 0.75 U ml^–1 were produced in the optimized culture medium supplemented with 1 mM MnSO₄ and 4 g l^–1 asparagine. The laccase titer under optimized conditions reached maximum values at 16 days of growth: 13.5 U ml^–1 in the presence of 0.2 mM CuSO₄, 0.4 mM MnSO₄ and 6 g l^–1 asparagine. Mn²⁺ promoted production of both enzymes. There were important interactions among the nutrients evaluated, the most significant being those between Cu²⁺ and asparagine.