Enhanced degradation and toxicity reduction of dihexyl phthalate by Fusarium oxysporum f. sp. pisi cutinase

AIMS: This research aims to investigate the efficiency of two lipolytic enzymes--fungal cutinase and yeast esterase--upon the biodegradation of dihexyl phthalate (DHP). METHOD AND RESULTS: During the enzymatic degradation of DHP dissolved in methanol, several degradation products were detected and their time-course changes were monitored using GC/MS. The DHP-degradation rate of cutinase was surprisingly high; i.e. almost 70% of the initial DHP (500 mg l(-1)) was decomposed within 4.5 h. Although the same amount of esterase was employed, more than 85% of the DHP remained after 3 days. Almost all the DHP was converted by cutinase into 1,3-isobenzofurandione (IBF), whereas hexyl methyl phthalate and IBF were abundantly produced by esterase. In addition, the toxicities of the DHP-degraded products by esterase were evaluated using various recombinant bioluminescent bacteria, which caused oxidative and protein damage, whereas the hydrolysis products from cutinase never caused any cellular damage in the methanol-containing reaction system. CONCLUSIONS: Cutinase starts to act as a DHP-degrader much earlier and faster than esterase, with high stability in ester-hydrolytic activity, therefore a plausible approach to the practical application of cutinase for DHP degradation in the DHP-contaminated environments may be possible. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the enhanced degradation and detoxification of DHP using Fusarium oxysporum f. sp. pisi cutinase.     

Biodegradation of dipropyl phthalate and toxicity of its degradation products: a comparison of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis> cutinase and <Emphasis Type="Italic">Candida cylindracea</Emphasis> esterase

The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of dipropyl phthalate (DPrP) was investigated. The DPrP-degradation rate of fungal cutinase was surprisingly high, i.e., almost 70% of the initial DPrP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DPrP disappeared within the initial 15 min. With the yeast esterase, despite the same concentration, more than 90% of the DPrP remained even after 3 days of treatment. During the enzymatic degradation of DPrP, several DPrP-derived compounds were detected and time-course changes in composition were also monitored. The final chemical composition after 3 days was significantly dependent on the enzyme used. During degradation with fungal cutinase, most DPrP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. However, in the degradation by yeast esterase, propyl methyl phthalate (PrMP) was produced in abundance in addition to IBF. The toxic effects of the final degradation products were investigated using various recombinant bioluminescent bacteria. As a result, the degradation products (including PrMP) from yeast esterase severely caused oxidative stress and damage to protein synthesis in bacterial cells, while in the fungal cutinase processes, DPrP was significantly degraded to non-toxic IBF after the extended period (3 days).     

Kinetics and pathway of biodegradation of dibutyl phthalate by Pleurotus ostreatus

Dibutyl phthalate (DBP) is a plasticizer, whose presence in the environment as a pollutant has attained a great deal of attention due to its reported association with endocrine system disturbances on animals. Growth parameters, glucose uptake, percentage of removal efficiency (%E) of DBP, biodegradation constant of DBP (k) and half-life of DBP biodegradation (t_1/2) were evaluated for Pleurotus ostreatus grown on media containing glucose and different concentrations of DBP (0, 500 and 1000 mg l^?1). P. ostreatus degraded 99.6 % and 94 % of 500 and 1000 mg of DBP l^?1 after 312 h and 504 h, respectively. The k was 0.0155 h^?1 and 0.0043 h^?1 for 500 and 1000 mg of DBP l^?1, respectively. t_1/2 was 44.7 h and 161 h for 500 and 1000 mg of DBP l^?1, respectively. Intermediate compounds of biodegraded DBP were identified by GC-MS and a DBP biodegradation pathway was proposed using quantum chemical calculation. DBP might be metabolized to benzene and acetyl acetate, the first would be oxidated to muconic acid and the latter would enter into the Krebs cycle. P. ostreatus has the ability to degrade DBP and utilizes it as source of carbon and energy.     

Fungal biodegradation of dibutyl phthalate and toxicity of its breakdown products on the basis of fungal and bacterial growth

Phthalates are esters of phthalic acid that give flexibility to polyvinyl chloride. Diverse studies have reported that these compounds might be carcinogenic, mutagenic and/or teratogenic. Radial growth rate, biomass, hyphal thickness of Neurospora sitophyla, Trichoderma harzianum and Aspergillus niger, grown in two different concentrations of dibutyl phthalate (DBP) (500 and 1,000 mg/l) in agar and in submerged fermentation were studied. The inhibitory concentration (IC₅₀) and the constant of biodegradation of dibutyl phthalate in Escherichia coli cultures were used to evaluate toxicity. The radial growth rate and thickness of the hypha were positively correlated with the concentration of phthalate. The pH of the cultures decreased as the fermentation proceeded. It is shown that these fungi are able to degrade DBP to non-toxic compounds and that these can be used as sole carbon and energy sources by this bacterium. It is demonstrated that the biodegradation of the DBP is directly correlated with the IC₅₀. This is the first study that reports a method to determine the biodegradation of DBP on the basis of the IC₅₀ and fungal growth, and the effect of this phthalate on the growth and thickness of hyphae of filamentous fungi in agar and in submerged fermentation.     

一株霍氏肠杆菌对四环素的降解作用及其降解产物的毒性评估

【目的】抗生素作为新兴污染物,已经引起社会的极大关注。针对四环素有效降解菌株缺乏这一现状,本研究旨在筛选和鉴定具有降解四环素功能的菌株,分析其降解特性和降解作用类型、初步探讨其降解活性物质定位并评估其降解产物的生理毒性。【方法】以四环素为唯一碳源,从受四环素污染的猪场污泥中筛选四环素降解菌株;结合菌落形态学特征、生理生化特征、扫描电镜观察和16S rRNA基因测序鉴定菌株,通过不同外源碳、pH及去除动力学阐明菌株对四环素的降解特性,提取菌株不同成分探讨其去除四环素的作用类型,并进一步从细胞内液和细胞外液开展生物降解的活性物质定位,最后评估降解产物的生理毒性。【结果】筛选鉴定得到一株霍氏肠杆菌(Enterobacter hormaechei) MEH2305,pH为7.0和添加10 g/L外源碳胰蛋白胨是其发挥降解作用的最适条件。MEH2305通过非生物降解和生物降解的共同作用,在培养第7天对四环素总去除率达到68% (对土霉素和盐酸强力霉素去除率分别为53%和56%),其分泌的细胞内液和细胞外液对四环素的去除率分别为40.77%和31.18%。同时,与未经MEH2305处理的四环素对照组比较,MEH2305降解四环素的产物对革兰氏阴性大肠杆菌(Escherichia coli) K88和革兰氏阳性枯草芽孢杆菌(Bacillus subtilis) 168的生理毒性作用显著降低。【结论】MEH2305可以作为一株潜在的有效且安全的四环素降解菌株,应用于抗生素的环境治理领域。     

Enhancement in the viability and biosensing activity of freeze-dried recombinant bioluminescent bacteria

The genetically-engineeredEscherichia coli strain, DPD2540, which contains afabA::luxCDABE fusion gene, gives a bioluminescent output when membrane fatty acid synthesis is needed. For more practical application of this strain in the field as biosensor, freeze-drying was adopted. A 12% sucrose solution with Luria-Bertani (LB) broth, as determined by the viability after freeze-drying, was found to be the most effective composition for lyophilization solution among various compositions tested. Rapid freezing with liquid nitrogen also gave the best viability after freeze-drying as compared to samples frozen at −70°C and −20°C. The biosensing activities of the cells showed a greater sensitivity when the cells from the exponential phase were freeze-dried. Finally, the optimum temperature for use of the freeze-dried cells in the biosensor field was determined.     

Toxicity monitoring of endocrine disrupting chemicals (EDCs) using freeze-dried recombinant bioluminescent bacteria

Five different freeze-dried recombinant bioluminescent bacteria were used for the detection of cellular stresses caused by endocrine disrupting chemicals. These strains were DPD2794 (recA::luxCDABE), which is sensitive to DNA damage, DPD2540 (fabA::luxCDABE), sensitive to cellular membrane damage, DPD2511 (katG::luxCDABE), sensitive to oxidative damage, and TV1061 (grpE::luxCDABE), sensitive to protein damage. GC2, which emits bioluminescence constitutively, was also used in this study. The toxicity of several chemicals was determined on the first four freeze-dried bacteria, while nonspecific cellular stresses were measured using GC2. Damage caused by known endocrine disrupting chemicals, such as nonyl phenol, bisphenol A, and styrene, was detected and classified according to toxicity mode, while others, such as phathalate and DDT, were not detected with the bacteria. These results suggest that endocrine disrupting chemicals are toxic in bacteria, and do not act via an estrogenic effect, and that toxicity monitoring and classification of some endocrine disrupting chemicals may be possible in the field using these freeze-dried recombinant bioluminescent bacteria.     

Contamination of Paddy Soil by Endocrine-Disrupting Chemicals Affects Soil Microbe Abundance and Diversity

We describe the effect on the population of Eubacteria and Archaea species of adding the endocrine-disrupting chemicals (EDCs) nonylphenol (NP) or dibutylphthalate (DBP) to a typical paddy soil. Fluorescence in-situ hybridization was used to discriminate between the two phyla, and denaturing gradient gel electrophoresis (DGGE) of an amplified fragment of the 16S rRNA locus was used to profile the species present. The population of both Eubacteria and Archaea species was reduced by the presence of NP or DBP, and the deleterious effect was greater for the Eubacteria. The DGGE profiles were used to assess the species diversity in the polluted and non-polluted soil samples. This showed that DBP was less damaging than NP₅₀. It was clear that EDCs can significantly affect paddy soil microbial diversity, both with respect to population size and species representation.     

Soil biosensor for the detection of PAH toxicity using an immobilized recombinant bacterium and a biosurfactant

A biosensor for detecting the toxicity of polycylic aromatic hydrocarbons (PAHs) contaminated soil has been successfully constructed using an immobilized recombinant bioluminescent bacterium, GC2 (lac::luxCDABE), which constitutively produces bioluminescence. The biosurfactant, rhamnolipids, was used to extract a model PAH, phenanthrene, and was found to enhance the bioavailability of phenanthrene via an increase in its rate of mass transfer from sorbed soil to the aqueous phase. The monitoring of phenanthrene toxicity was achieved through the measurement of the decrease in bioluminescence when a sample extracted with the biosurfactant was injected into the minibioreactor. The concentrations of phenanthrene in the aqueous phase were found to correlate well with the corresponding toxicity data obtained by using this toxicity biosensor. In addition, it was also found that the addition of glass beads to the agar media enhanced the stability of the immobilized cells. This biosensor system using a biosurfactant may be applied as an in-situ biosensor to detect the toxicity of hydrophobic contaminants in soils and for performance evaluation of PAH degradation in soils.     

Reproductive toxicity and pharmacokinetics of di-n-butyl phthalate (DBP) following dietary exposure of pregnant rats

Most rodent developmental toxicity studies of dibutylphthalate (DBP) have relied on bolus gavage dosing. This study characterized the developmental toxicity of dietary DBP. Pregnant CD rats were given nominal doses of 0, 100, or 500 mg DBP/kg/day in diet (actual intake 0, 112, and 582 mg/kg/day) from gestational day (GD) 12 through the morning of GD 19. Rats were killed 4 or 24 hr thereafter. DBP dietary exposure resulted in significant dose-dependent reductions in testicular mRNA concentration of scavenger receptor class B, member 1; steroidogenic acute regulatory protein; cytochrome P450, family 11, subfamily a, polypeptide 1; and cytochrome P450 family 17, subfamily a, polypeptide 1. These effects were most pronounced 4 hr after the end of exposure. Testicular testosterone was reduced 24 hr post-exposure in both DBP dose groups and 4 hr after termination of the 500-mg DBP/kg/day exposure. Maternal exposure to 500 mg DBP/kg/day induced a significant reduction in male offspring's anogenital distance indicating in utero disruption of androgen function. Leydig cell aggregates, increased cord diameters, and multinucleated gonocytes were present in DBP-treated rats. Monobutyl phthalate, the developmentally toxic metabolite of DBP, and its glucuronide conjugate were found in maternal and fetal plasma, amniotic fluid, and maternal urine. Our results, when compared to previously conducted gavage studies, indicate that approximately equal doses of oral DBP exposure of pregnant rats, from diet or gavage, result in similar responses in male offspring. Birth Defects Res (Part B), 86:345–354, 2009. © 2009 Wiley-Liss, Inc.     

Biodegradation of diethyl phthalate by an organic-solvent-tolerant Bacillus subtilis strain 3C3 and effect of phthalate ester coexistence

The contamination and distribution of phthalate esters - synthetic compounds widely used in plastic product production, including food and medical packaging - has raised safety concerns due to their endocrine-disrupting activity and mandated to be treated. Bacillus subtilis strain 3C3, isolated as an organic-solvent-tolerant bacterium, was capable of utilizing diethyl phthalate as a sole carbon source. Biodegradation of diethyl phthalate occurred constitutively without lag period, and its kinetics followed a first-order model. The biodegradability was significantly enhanced with the supplementation of yeast extract as a co-metabolic substrate. In the presence of Tween-80 as a solubilizing agent, cells rapidly degrade a range of short-chain phthalate esters at high concentrations (up to 1000 mg l⁻¹ for diethyl phthalate). The biodegradation of short-chain phthalates in the binary, ternary and quaternary substrate system revealed that the coexistence of other short-chain phthalates had no significant influence on the biodegradation of diethyl phthalate, and vice versa. These results substantiated that B. subtilis strain 3C3 has potential application as a bioaugmented bacterial culture for bioremediation of phthalates.     

Enzymatic removal of bilirubin toxicity by bilirubin oxidase in vitro and excretion of degradation products in vivo

The toxic effects of the degradation products of bilirubin that were formed by reaction with bilirubin oxidase were investigated with the C 1300 mouse neuroblastoma cell line by examining the following parameters: growth inhibition, morphologic characteristics, membrane transport, DNA synthesis, and protein synthesis. The addition of bilirubin to the cells resulted in definite cytotoxic effects on all of these parameters in a dose-dependent fashion; the addition of bilirubin oxidase reversed the toxic effects on the C 1300 cells in vitro. Furthermore, we found that most of these enzymatic degradation products of bilirubin were excreted by the kidney into the urine in a few hours after intravenous injection of the degradation products; in contrast, no intact bilirubin was excreted. Thus, these findings suggest that hyperbilirubinemia in newborn infants (kernicterus) may be prevented by administering polyethylene glycol-conjugated bilirubin oxidase, with a longer plasma half-life which has been reported previously to oxidize bilirubin to its nontoxic components in the bloodstream.     

Acclimation and repair of DNA damage in recombinant bioluminescent Escherichia coli

AIMS: The aim of this study is to understand different adaptive responses in bacteria caused by three different mutagens, namely, an intercalating agent, an alkylating agent and a hydroxylating agent, and the repair systems according to the type of DNA damage, that is, DNA cross-linking and delayed DNA synthesis, alkylation and hydroxylation of DNA. A recombinant bioluminescent Escherichia coli, DPD2794 with the recA promoter fused to luxCDABE originating from Vibrio fischeri, was used in this study. METHODS AND RESULTS: The recombinant bioluminescent E. coli strain DPD2794, containing a recA promoter fused to luxCDABE from V. fischeri, was used to detect adaptive and repair responses to DNA damage caused by mitomycin C (MMC), and these responses were compared with those when the cells were induced with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2). The response ratio between the induced samples and that of the controls decreased suddenly when the induced culture was used in further inductions, indicating a possible adaptive response to DNA damage. DNA damage, or the proteins produced, because of MMC addition does not appear to be completely resolved until the seventh sub-culture after the initial induction, whereas simple damage, such as the base modification caused by MNNG and H2O2, appears to be repaired rapidly as evidenced by the quick recovery of sensitivity. CONCLUSIONS: These results suggest that it takes more time to completely repair DNA damage caused by MMC, as compared with a simple repair such as that required for the damage caused by MNNG and H2O2. Therefore, repair of the damage caused by these three mutagens is controlled by different regulons, even though they all induced the recA promoter. SIGNIFICANCE AND IMPACT OF THE STUDY: Using a bioluminescent E. coli harbouring a recA promoter-lux fusion, it was found that different adaptive responses and repair systems for DNA damage caused by several mutagens exists in E. coli.     

土壤中多环芳烃的微生物降解及土壤细菌种群多样性

利用室内模拟方法,研究中、低浓度多环芳烃(PAHs)污染土壤的微生物修复效果,阐明土壤微生物（接种和土著）与PAHs降解的关系.结果表明:投加PAHs高效降解菌可以促进土壤中PAHs的降解,2周内效果显著;典型PAHs降解的难易程度依据为：菲<蒽<芘<苯并(a)芘和屈;细菌种群丰度和多样性均与PAHs降解呈负相关关系,同一处理细菌种群结构随时间变化不大.对于中、低浓度PAHs原位污染土壤,增强土著菌的活性是提高土壤PAHs降解率的有效途径之一.     

几种杀螺细菌的筛选与功效研究

湖北钉螺是日本血吸虫的唯一中间宿主,生物杀螺具有环保、成本较低以及对钉螺定向特异的优点。从鄱阳湖草洲土壤及病螺体内分离出4株对钉螺具有高毒性作用的细菌。革兰氏染色和显微镜观察表明,一株为芽孢杆菌,其余3株均为革兰氏阴性杆菌。16srDNA扩增及序列比对鉴定其种属类型,结果显示分别为Enterobactersp．、B．cereus、S．putrefaciens和Aeromonassp．。杀螺研究结果表明,4株细菌的发酵液、发酵上清液和菌体对钉螺均表现出不同程度的毒杀作用,其中S．putrefaciens和Aeromonassp．的杀螺效果最好,72h发酵液和发酵上清液对钉螺的毒杀作用分别达100％和95％以上。各细菌发酵上清SDS-PAGE结果发现了丰富蛋白带,推测这些蛋白中存在对钉螺具有毒杀功效的组分。     

Isolation from coastal sea water and characterization of bacterial strains involved in non-ionic surfactant degradation

A bacterial community degrading branched alkylphenol ethoxylate (APE) was selected from coastal sea water intermittently polluted by urban sewage. This community degraded more than 99% of a standard surfactant, TRITON X 100, but I.R. analysis of the remaining compound showed the accumulation of APE₂ (alkylphenol with a two units length ethoxylated chain) which seemed very recalcitrant to further biodegradation. Twenty-five strains were isolated from this community, essentially Gram negative and were related to Pseudomonas, Oceanospirillum or Deleya genera. Among these strains, only four were able to degrade APE_9–10 (TRITON X 100). They were related to the Pseudomonas genus and were of marine origin. Pure cultures performed with these strains on TRITON X 100 gave APE₅ and APE₄ as end products. These products were further degraded to APE2 by two other strains unable to degrade the initial surfactant.     

Decoloration of textile dyes by Trametes versicolor and its effect on dye toxicity

Amaranth, Tropaeolin O, Reactive Blue 15, Congo Red, and Reactive Black 5 were completely decolorized with no dye sorption by Trametes versicolor. Cibacron Brilliant Red 3G-P, Cibacron Brilliant Yellow 3B-A, and Remazol Brilliant Blue R were partially decolorized with some dye sorbed to the biomass. The Microtox assay before decoloration showed that Amaranth and Tropaeolin O were not toxic [the percent concentration to decrease 20% of the luminescence of Vibrio fischeri (EC₂₀) was greater than 100%]; Cibacron Brilliant Yellow 3B-A, Reactive Blue 15 and Cibacron Brilliant Red 3G-P were moderately non-toxic (100% > EC₂₀ > 75%); Remazol Brilliant Blue R was toxic (75% > EC₂₀ > 50%); and Congo Red and Reactive Black 5 were moderately toxic (50% > EC₂₀ > 25%). After decoloration the toxicity of the solutions containing Amaranth, Tropaeolin O and Reactive Black 5 was unchanged; Reactive Blue 15, Remazol Brilliant Blue R and Cibacron Brilliant Red 3G-P decreased to non-toxic levels; and Cibacron Brilliant Yellow 3B-A and Congo Red became very toxic (EC₂₀ < 25%).