Microbial detoxification of metals and radionuclides

Microorganisms have important roles in the biogeochemical cycling of toxic metals and radionuclides. Recent advances have been made in understanding metal-microbe interactions and new applications of these processes to the detoxification of metal and radionuclide contamination have been developed.     

Dual effects of phloretin on aflatoxin B1 metabolism: activation and detoxification of aflatoxin B1

Typically, chemopreventive agents involve either induction of phase II detoxifying enzymes and/or inhibition of cytochrome P450 enzymes (CYPs) that are required for the activation of procarcinogens. In this study, we investigated the protective effects of phloretin against aflatoxin B1 (AFB1) activation to the ultimate carcinogenic intermediate, AFB(1)-8, 9-epoxide (AFBO), and its subsequent detoxification. Phloretin markedly inhibited formation of the epoxide with human liver microsomes in a dose-dependent manner. Phloretin also inhibited the activities of nifedipine oxidation and ethoxyresorufin O-deethylase (EROD) in human liver microsomes. These data show that phloretin strongly inhibits CYP1A2 and CYP3A4 activities, which are involved in the activation of AFB1. Phloretin increased glutathione S-transferase (GST) activity of alpha mouse liver 12 (AML 12) cells in a dose-dependent manner. GST activity toward AFBO in cell lysates treated with 20 μM phloretin was 23-fold that of untreated control cell lysates. The expression of GSTA3, GSTA4, GSTM1, GSTP1 and GSTT1 was induced by phloretin in a dose-dependent manner in AML 12 cells. GSTP1, GSTM1, and GSTT1 were able to significantly increase the conjugation of AFBO with glutathione. Concurrently, induction of the GST isozyme genes was partially associated with the Nrf2/ARE pathway. Taken together, the results demonstrate that phloretin has a strong chemopreventive effect against AFB1 through its inhibitory effect on CYP1A2, CYP3A4, and its inductive effect on GST activity.     

Microbial detoxification of waste rubber material by wood-rotting fungi

The extensive use of rubber products, mainly tires, and the difficulties to recycle those products, has resulted in world wide environmental problems. Microbial devulcanisation is a promising way to increase the recycling of rubber materials. One obstacle is that several microorganisms tested for devulcanisation are sensitive to rubber additives. A way to overcome this might be to detoxify the rubber material with fungi prior to the devulcanisation. In this study, 15 species of white-rot and brown-rot fungi have been screened with regard to their capacity to degrade an aromatic model compound in the presence of ground waste tire rubber. The most effective fungus, Resinicium bicolor, was used for detoxification of rubber material. Increase in growth of the desulfurising bacterium Thiobacillus ferrooxidans in presence of the rubber treated with Resinicium bicolor compared to untreated rubber demonstrated that detoxification with fungi is possible.     

Microbial metabolism and detoxification of 7,12-dimethylbenz[a]anthracene

Summary Six strains of fungi grown on Sabouraud dextrose broth in the presence of 7,12-dimethylbenz[a]anthracene (DMBA) were surveyed for their ability to metabolize DMBA. Experiments with [¹⁴C]DMBA indicated that the extent of formation of organic-soluble metabolites ranged from 6 to 28% after 5 days of incubation, depending on the organism tested. The yields of water-soluble metabolites also varied, and ranged from 1 to 33% after 5 days.Cunninghamella elegans ATCC 36112 andSyncephalastrum racemosum UT-70 exhibited the highest DMBA-metabolizing activity among the organisms surveyed.S. racemosum metabolized DMBA primarily to 7-hydroxymethyl-12-methylbenz[a]anthracene (7-OHM-12-MBA)_ and 7,12-dihydroxymethylbenz[a]anthracene (7,12-diOHMBA). Minor metabolites included 7-OHM-12-MBA-trans-5,6-, 8,9- and 10,11-dihydrodiols, and glucuronide and sulfate conjugates of phenolic derivatives of DMBA. In contrast, the major DMBA metabolites produced byC. elegans were water-soluble. The predominant organic-soluble metabolites produced byC. elegans included 7-OHM-12-MBA-trans-5,6-, 8,9- and 10,11-dihydrodiols. DMBA-trans-3,4-dihydrodiol was also detected. Circular dichroism spectral analysis revealed that the major enantiomer of the 7-OHM-12-MBA-trans-8,9-dihydrodiol formed by each organism has anS,S absolute configuration, while the major enantiomers of the 5,6-, 10,11- and 3,4-dihydrodiols had anR,R configuration. The mutagenic activity of extracts fromS. racemosum exposed to DMBA were determined inSalmonella typhimurium TA98. The mutagenicity of DMBA decreased by 36% over a period of 5 days as 33% of the compound was metabolized. Comparison of these results with previously reported results in mammalian systems suggests that there are similarities and differences between the fungal and mammalian oxidation of DMBA and that the overall balance of fungal metabolism is towards a detoxification rather than a bioactivation pathway.     

A pilot plant for detoxification of aflatoxin B1-contaminated coconut oil by solar irradiation

Summary In coconut oil naturally contaminated with aflatoxin B1, more than 85% of the toxin is present in the soluble form, the remainder occuring in the sediment. This aflatoxin is detoxified when the oil, in a static layer less than 15 mm thick is exposed to solar radiation. A pilot plant, designed to take account of the viscosity and flow characteristics of the oil, was constructed for the exposure of thin layers of oil (2 mm or less) flowing under gravity. At aflatoxin concentrations between 166 and 1250 jug/kg, 75% of the toxin was degraded on exposure to solar radiation of 10 cal/cm²; total detoxification was achieved on repeated exposure. The naturally contaminated coconut oil after exposure to solar radiation did not contain any residual aflatoxins or fluorescent compounds which might have been derived from original aflatoxin B1.

---

Resumen Planta pilota para el tratramiento de aceite de coco contaminado con aflatoxina B1 mediante radiation solar y centrifugado En aceite de coco contaminado de forma natural con aflatoxina B1, más del 85% de la toxina se encuentra en forma soluble, el resto permaneciendo en el sedimento. Esta aflatoxina pierde sus propiedades tóxicas cuando se expone el aceite en forma de capa estática de 15 mm de grosor a la radiación solar. Se construyó una planta piloto diseñada teniendo en cuenta la viscosidad y las caracteristicas de fluidez del aceite de forma que pudieran exponerse a la radiación solar capas de aceite muy finas (2 mm o menos) fluyendo gracias a la gravedad. Cuando aceite conteniendo aflatoxina en proporciones entre 166 y 1250 /kg se expuso a una radiación solar de 10 cal./cm² la toxina se degradó en un 75%. La detoxificación total se obtuvo mediante repetición del proceso. El aceite de coco contaminado de forma natural, una vez expuesto a la radiación solar no contenía aflatoxinas residuales ni compuestos fluorescentes potencialmente derivados de la aflatoxina B1 contenida originalmente.

---

Résumé Usine-pilote pour la détoxification, par irradiation solaire et centrifugation, de l'huile de coprah contaminée par l'aflatoxine B1 Dans l'huile de coprah spontanément contaminée par l'aflatoxine B1, plus de 85% de la toxine est présente sous forme soluble, le reste se trouvant dans le sédiment. Cette aflatoxine est détoxifiée lorsque l'huile est exposée à la radiation solaire en couche statique de moins de 15 mm d'épaisseur. Une usine-pilote, conçue en tenant compte de la viscosité et de l'écoulement de l'huile, a été construite pour irradier une mince couche d'huile (2 mm ou moins) s'écoulant par gravité. Pour des concentrations en aflatoxine allant de 166 à 1250 g/kg, 75% de la toxine est dégradée par exposition à une irradiation solaire de 10 cal./cm² et, en répétant le traitement, on obtient une détoxification complète. Après exposition à la radiation solaire, l'huile de coprah contaminée spontanément ne contient plus d'aflatoxine résiduelle, ni de composés fluorescents dérivés de l'aflatoxine B1 originelle.

     

Microbial degradation and detoxification of 2,4-dinitrophenol in aerobic and anoxic processes

A bacterial strain was isolated from a river sediment in Buenos Aires, Argentina, owing to its ability to utilize 2,4-dinitrophenol (2,4-DNP) as the sole carbon, nitrogen and energy source. The strain was identified as Rhodococcus opacus on the basis of its 16S rRNA gene sequence. R. opacus degrades aerobically 0.27 and 0.54 mM within 22 and 28 h, respectively, and releases the nitro groups from 2,4-DNP as nitrites. Aerobic biodegradation processes were performed using a 2-l volume microfermentor at with agitation (200 rpm), and were evaluated by spectrophotometry, high performance liquid chromatography (HPLC) and microbial growth. The absence of 2,4-DNP transformation products was also confirmed by gas chromatography mass spectrometry (GC–MS). As the nitrite released during 2,4-DNP degradation is in addition an environmental toxic agent it was removed by denitrification in an anoxic process. Detoxification was assessed by using luminescent bacteria, algae and seeds toxicity tests. Toxicity was not detected after combining both the aerobic and anoxic processes.     

Microbial detoxification of pathotoxin produced by spot blotch pathogen Bipolaris sorokiniana infecting wheat

Bipolaris sorokiniana causes spot blotch in wheat and barley. The pathogen produces toxin (BS-toxin), which is a sesquiterpenoid belonging to eremophilane family. Isolates of Trichoderma spp., Chaetomium globosum and Pseudomonas fluorescens were tested for detoxification of BS-toxin amended in semi-synthetic medium at different concentrations. All the antagonists showed mycelial growth in toxin amended medium but their growth was less in comparison to growth in normal medium. The growth of biocontrol agents decreased with increasing concentration of toxin. Two isolates of C. globosum (Cg1 and Cg2), T.viride (TV5-2) and Pseudomonas fluorescens produced 4.9, 2.9, 3.6 g mycelium and 5.5 × 10⁵ cfu /ml, respectively exhibiting 50% or less reduction in growth in BS-toxin amended medium at 1,000 ppm concentration. The biocontrol agents also reduced the severity of toxin-induced symptoms and electrolyte leakage from the wheat leaf tissues. Among the microbes tested, maximum reduction in electrolyte leakage was observed in C. globosum (Cg2) treated toxin samples. The spectral analysis also showed a remarkable decrease in optical density of Cg2 treated toxin at 294 nm. High Performance Liquid Chromatography (HPLC) analysis showed almost complete degradation of BS-toxin in C. globosum (Cg2) treated samples.     

Microbial detoxification of bifenthrin by a novel yeast and its potential for contaminated soils treatment

Bifenthrin is one the most widespread pollutants and has caused potential effect on aquatic life and human health, yet little is known about microbial degradation in contaminated regions. A novel yeast strain ZS-02, isolated from activated sludge and identified as Candida pelliculosa based on morphology, API test and 18S rDNA gene analysis, was found highly effective in degrading bifenthrin over a wide range of temperatures (20-40 °C) and pH (5-9). On the basis of response surface methodology (RSM), the optimal degradation conditions were determined to be 32.3 °C and pH 7.2. Under these conditions, the yeast completely metabolized bifenthrin (50 mg · L(-1)) within 8 days. This strain utilized bifenthrin as the sole carbon source for growth as well as co-metabolized it in the presence of glucose, and tolerated concentrations as high as 600 mg · L(-1) with a q(max), K(s) and K(i) of 1.7015 day(-1), 86.2259 mg · L(-1) and 187.2340 mg · L(-1), respectively. The yeast first degraded bifenthrin by hydrolysis of the carboxylester linkage to produce cyclopropanecarboxylic acid and 2-methyl-3-biphenylyl methanol. Subsequently, 2-methyl-3-biphenylyl methanol was further transformed by biphenyl cleavage to form 4-trifluoromethoxy phenol, 2-chloro-6-fluoro benzylalcohol, and 3,5-dimethoxy phenol, resulting in its detoxification. Eventually, no persistent accumulative product was detected by gas chromatopraphy-mass spectrometry (GC-MS) analysis. This is the first report of a novel pathway of degradation of bifenthrin by hydrolysis of ester linkage and cleavage of biphenyl in a microorganism. Furthermore, strain ZS-02 degraded a variety of pyrethroids including bifenthrin, cyfluthrin, deltamethrin, fenvalerate, cypermethrin, and fenpropathrin. In different contaminated soils introduced with strain ZS-02, 65-75% of the 50 mg · kg(-1) bifenthrin was eliminated within 10 days, suggesting the yeast could be a promising candidate for remediation of environments affected by bifenthrin. Finally, this is the first described yeast capable of degrading bifenthrin.     

Microbial degradation and detoxification of high molecular weight polycyclic aromatic hydrocarbons by Stenotrophomonas maltophilia strain VUN 10,003

The ability of Stenotrophomonas maltophilia strain VUN 10,003 to degrade and detoxify high molecular weight polycyclic aromatic hydrocarbons (PAHs) was evaluated in a basal liquid medium. Using high cell density inocula of strain VUN 10,003, the concentration of pyrene, fluoranthene, benz[a]anthracene, benzo[a]pyrene, dibenz[a, h]anthracene and coronene decreased by 98, 45, 26, 22, 22 and 55% over periods ranging from 5 to 42 d. When a PAH mixture containing three- to seven-ring compounds was used, degradation of both low and high molecular weight compounds occurred concurrently. Mutagenicity assays (Ames Test) demonstrated a decrease in the mutagenic potential of dichloromethane culture extracts from all cultures containing single PAH over the incubation period, corresponding to the decrease in the concentration of the PAH. These observations indicate that strain VUN 10,003 could be used for the detoxification of PAH-contaminated wastes.     

Isolation of Bacillus spp. from Thai fermented soybean (Thua-nao): screening for aflatoxin B1 and ochratoxin A detoxification

Aims: To study the interaction between Bacillus spp. and contaminating Aspergillus flavus isolated strains from Thai fermented soybean in order to limit aflatoxin production. To study the detoxification of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) by Bacillus spp. in order to find an efficient strain to remove these toxins. Methods and Results: One A. flavus aflatoxin-producing strain and 23 isolates of Bacillus spp. were isolated from soybean and fresh Thua-nao collected from the north of Thailand. Inhibition studies of A. flavus and A. westerdijkiae NRRL 3174 (reference strain) growth by all isolates of Bacillus spp. were conducted by dual culture technique on agar plates. These isolates were also tested for AFB₁ and OTA detoxification ability on both solid and liquid media. Most of the strains were able to detoxify aflatoxin but only some of them could detoxify OTA. Conclusions: One Bacillus strain was able to inhibit growth of both Aspergillus strains and to remove both mycotoxins (decrease of 74% of AFB₁ and 92·5% of OTA). It was identified by ITS sequencing as Bacillus licheniformis. The OTA decrease was due to degradation in OTα. Another Bacillus strain inhibiting both Aspergillus growth and detoxifying 85% of AFB₁ was identified as B. subtilis. AFB₁ decrease has not been correlated to appearance of a degradation product. Significance and Impact of the Study: The possibility to reduce AFB₁ level by a strain from the natural flora is of great interest for the control of the quality of fermented soybean. Moreover, the same strain could be a source of efficient enzyme for OTA degradation in other food or feeds.     

Ecology of aflatoxin producing fungi and biocontrol of aflatoxin contamination

Summary  Aflatoxins, highly toxic and carcinogenic compounds that frequently contaminate foods and feeds, are produced by several genera in the genusAspergillus. Aspergillus flavus, the most common species causing crop contamination, is a common inhabitant of the Sonoran desert of North America where it resides in complex communities composed of diverse individuals. This diversity reflects divergent adaptation to various ecological niches. SomeA. flavus isolates that are well adapted to plant associated niches do not produce aflatoxins yet have the capacity to competitively exclude aflatoxin producers. These atoxigenic strains can serve as biological control agents for management of aflatoxins in crops. Detailed knowledge of the ecology of aflatoxin-producing fungi may lead to novel practical methods for limiting contamination. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005.     

Biochemistry of arsenic detoxification

All living organisms have systems for arsenic detoxification. The common themes are (a) uptake of As(V) in the form of arsenate by phosphate transporters, (b) uptake of As(III) in the form of arsenite by aquaglyceroporins, (c) reduction of As(V) to As(III) by arsenate reductases, and (d) extrusion or sequestration of As(III). While the overall schemes for arsenic resistance are similar in prokaryotes and eukaryotes, some of the specific proteins are the products of separate evolutionary pathways.     

Comparison of urinary aflatoxin M1 and aflatoxin albumin adducts as biomarkers for assessing aflatoxin exposure in Tanzanian children

Purpose: To determine levels of urinary aflatoxin M1 (AFM1) in children and correlate the concentrations with previously reported aflatoxin albumin adduct (AF-alb) levels in these children.

Materials and methods: Matched urine and blood samples were collected from 84 Tanzanian children aged 6–14 months old. From 31 children in one village (Kigwa), samples were collected at three time points six months apart. Samples were collected from 31 and 22 children from two different regions at the second time point only. Urinary AFM1 was measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit with a modified protocol to improve sensitivity. AF-alb was measured using an established ELISA method.

Results: The relative ranking of the three villages for exposure to aflatoxin based on either AFM₁ or AF-alb biomarker measurements was the same. In Kigwa village, both AFM1 and AF-alb levels were higher at six months post-harvest compared to baseline. However, at the next visit, the AFM1 levels dropped from a GM (interquartile range) of 71.0 (44.7, 112.6) at visit two to 49.3 (31.5, 77.3) pg/ml urine, whereas AF-alb levels increased from 47.3 (29.7, 75.2) to 52.7 (35.4, 78.3) pg/mg albumin between these two visits, reflecting the fact that AFM1 measures short-term exposure, whereas AF-alb measures longer term exposure. There was a correlation between AFB1 intake and AFM1 excretion (r=?0.442, p?≤?0.001).

Conclusions: Urinary AFM1 is a good biomarker for AFB1 exposure in Tanzanian children, reflecting geographical and temporal variations in exposure to this foodborne toxin.     

Laccase-mediated detoxification of phenolic compounds

The ability of a polyphenoloxidase, the laccase of the fungus Rhizoctonia praticola, to detoxify phenolic pollutants was examined. The growth of the fungus could be inhibited by phenolic compounds, and the effective concentration was dependent on the substituents of the phenol. A toxic amount of a phenolic compound was added to a fungal growth medium in the presence or absence of a naturally occurring phenol, and half of the replicates also received laccase. The medium was then inoculated with R. praticola, and the levels of phenols in the medium were monitored by high-performance liquid chromatography analysis. The addition of the laccase reversed the inhibitory effect of 2,6-xylenol, 4-chloro-2-methylphenol, and p-cresol. Other compounds, e.g., o-cresol and 2,4-dichlorophenol, were detoxified only when laccase was used in conjunction with a natural phenol such as syringic acid. The toxicity of p-chlorophenol and 2,4,5-trichlorophenol could not be overcome by any additions. The ability of the laccase to alter the toxicity of the phenols appeared to be related to the capacity of the enzyme to decrease the levels of the parent compound by transformation or cross-coupling with another phenol.     

Bacterial detoxification of diisopropyl fluorophosphate

The ability of 18 gram-negative bacterial isolates to detoxify diisopropyl fluorophosphate, a structural analog of the agents soman and sarin, was investigated. Detoxification by both frozen cell sonicates and acetone powders was assayed by two methods, i.e., the hydrolytic release of fluoride, measured by a fluoride-specific ion electrode, and the disappearance of acetylcholinesterase inhibition in vitro. Frozen cell sonicates for all strains exhibited some activity (F- ion release). In general, acetone powder preparations produced higher activity than frozen cell sonicates did, and the highest activities were exhibited by strains with known parathion hydrolase activity. Two ranges in activity were observed, low level, ranging from 0.1 to 7.0 mumol/min per g of protein, and high level, detected only in parathion hydrolase-producing strains, from 47 to greater than 300 mumol/min per g of protein. Results indicate that parathion hydrolase was nonspecific in phosphoesterase activity. Also, it was an effective detoxicant at low concentrations and near-neutral pH.