Biodegradation of diethyl phthalate in soil by a novel pathway

Biodegradation of diethyl phthalate (DEP) has been shown to occur as a series of sequential steps common to the degradation of all phthalates. Primary degradation of DEP to phthalic acid (PA) has been reported to involve the hydrolysis of each of the two diethyl chains of the phthalate to produce the monoester monoethyl phthalate (MEP) and then PA. However, in soil co-contaminated with DEP and MeOH, biodegradation of the phthalate to PA resulted in the formation of three compounds, in addition to MEP. These were characterised by gas chromatography-electron ionisation mass spectrometry and nuclear magnetic resonance as ethyl methyl phthalate, dimethyl phthalate and monomethyl phthalate, and indicated the existence of an alternative pathway for the degradation of DEP in soil co-contaminated with MeOH. Transesterification or demethylation were proposed as the mechanisms for the formation of the three compounds, although the 7:1 ratio of H(2)O to MeOH means that transesterification is unlikely.     

Biodegradation of phthalic acid esters (PAEs) in soil bioaugmented with acclimated activated sludge

The degradation characteristics of four phthalic acid esters (PAEs), i.e. di-methyl phthalate (DMP), di-ethyl phthalate (DEP), di-n-butyl phthalate (DBP) and di-n-octyl phthalate (DOP) in the soil augmented with acclimated sludge was investigated in order to assess the efficacy of bioaugmentation as a strategy for remediating PAEs-contaminated soil and correlate the degradation rate of PAEs with their alkyl chain length. The results demonstrated that PAEs with shorter alkyl chain, that is, DMP and DEP could be degraded more quickly than DBP and DOP. The degradation of four PAEs in the soil conformed to a first-order reaction kinetic equation. The half-lives of PAEs degradation decreased significantly with increasing carbon number of the alcohol moiety. Half-lives decreased from 2.29 days for DMP to 28.4 days for DOP when the carbon number of alkyl chain increased from one for DMP to eight for DOP. The degradation rate of PAEs and the corresponding half-lives could correlate with the alkyl chain length and their octanol–water partition coefficients (K_ow) quite well for the four PAEs tested in this study.     

Kinetic analysis of the transformation of phthalate esters in a series of stoichiometric reactions in anaerobic wastes

Phthalates such as dimethyl phthalate, dimethyl terephthalate (DMT), diethyl phthalate (DEP), di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate (MEHP) are degraded to varying degrees under anaerobic conditions in waste treatment systems. Here we kinetically analyse the enzymatic hydrolyses involved and the subsequent stoichiometric reactions. The resulting model indicates that the degradation of the alcohols released and the transformation of the phthalic acid (PA) result in biphasic kinetics for the methane formation during transformation of DMT, DEP and MEHP. The ester hydrolysis and the PA transformation to methane appear to be the two rate-limiting steps. The PA-fermenting bacteria, which have biomass-specific growth rates between 0.04 and 0.085 day⁻¹, grow more slowly than the other bacteria involved. Anaerobic microorganisms that remove intermediate products during phthalic acid ester conversion appear to be important for the efficiency of the ultimate phthalate degradation and to be inhibited by elevated hydrogen partial pressures. The model was based on (and the simulations corresponded well with) data obtained from experimental waste treatment systems.     

Dibutyl phthalate biodegradation by the white rot fungus, Polyporus brumalis

In this study, white rot fungus, Polyporus brumalis, was applied to degrade dibutyl phthalate (DBP), a major environmental pollutant. The degradation potential and resulting products were evaluated with HPLC and GC/MS. As DBP concentration increased to 250, 750, and 1,250 microM, the mycelial growth of P. brumalis was inhibited. However, growth was still observed in the 1,250 microM concentration. DBP was nearly eliminated from culture medium of P. brumalis within 12 days, with 50% of DBP adsorbed by the mycelium. Diethyl phthalate (DEP) and monobutyl phthalate (MBP) were detected as intermediate degradation products of DBP. In culture medium, the concentration of DEP was higher than that of MBP during the incubation period. After 12-15 days, the concentrations of both decreased rapidly in the culture medium. The primary final degradation product of DBP in culture medium was phthalic acid anhydride, as well as trace amounts of aromatic compounds, such as alpha-hydroxyphenylacetic acid, benzyl alcohol, and O-hydroxyphenylacetic acid. According to these results, the degradation of DBP in culture medium by the white rot fungus, P. brumalis, may be completed through two pathways-transesterification and de-esterification-which successively combine into an intracellular degradation pathway.     

Isolation and characterization of four di-<Emphasis Type="Italic">n</Emphasis>-butyl phthalate (DBP)-degrading <Emphasis Type="Italic">Gordonia</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> strains and cloning the 3,4-phthalate dioxygenase gene

Four aerobic bacterial strains capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy were isolated from river sediments. Based on the morphology, biochemical characterization, and 16S rRNA gene sequence analysis, they were identified as Gordonia sp. The optimal conditions for DBP degradation by these strains were found to be pH 7.0, 30°C, and stirring at 175 rpm. These four strains could degrade, respectively, 96, 98, 98, and 78% of DBP (400 mg l⁻¹) as well as dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-octyl phthalate (DOP), di-isooctyl phthalate (DIOP), and di-isononyl phthalate (DINP). Furthermore, partial sequences of the gene for 3,4-phthalate dioxygenase were obtained from all four strains. To our knowledge, this is the first time that the 3,4-phthalate dioxygenase gene has been successfully cloned from Gordonia sp.     

城市污泥与稻草堆肥中邻苯二甲酸酯（PAEs）的研究

将广州城市污泥与稻草进行翻堆、接菌-翻堆、连续通气和间歇通气4种方式的堆肥,应用GC／MS技术对堆肥中6种属于USEPA优控污染物的邻苯二甲酸醇化合物(PAEs)进行分析,探讨堆肥产物中PAEs的含量分布以及不同方式堆肥对PAEs的降解效果,结果表明,4种方式堆肥中PAEs总含量(∑PAEs)在9．815～17．832mg·kg^-1之间,依次为翻堆(17．832mg·kg^-1)>接菌-翻堆(13．927mg·kg^-1)>间隙通气(10．765mg·kg^-1)>连续通气(9．815mg·kg^-1),堆肥中PAEs以邻苯二甲酸正二辛酯(DhOP)为主,占∑PAEs的82．2％～89．696,不同方式堆肥中∑PAEs的降解率为连续通气(45．71％)>间隙通气(40．4696)>接菌-翻堆(22．97％)>翻堆(1．3796)(平均降解率为27．63％),其中邻苯二甲酸二乙醇(DEP)、邻苯二甲酸正二丁酯(DnBP)和邻苯二甲酸丁基苄基酯(BBP)的降解率分别为95．7696～98．6896、79．5696～99．46％和87．42％～98．42％;但邻苯二甲酸二甲酯(DMP)和邻苯二甲酸正二辛酯的含量反而增加,邻苯二甲酸(2-乙基己基)酯(DEHP)在所有堆肥中均未检出。     

Thermal and enzymatic pretreatment of sludge containing phthalate esters prior to mesophilic anaerobic digestion

The present study aimed at investigating the effect of thermal pretreatment of sludge at 70 degrees C on the anaerobic degradation of three commonly found phthalic acid esters (PAE): di-ethyl phthalate (DEP), di-butyl phthalate (DBP), and di-ethylhexyl phthalate (DEHP). Also, the enzymatic treatment at 28 degrees C with a commercial lipase was studied as a way to enhance PAE removal. Pretreatment at 70 degrees C of the sludge containing PAE negatively influenced the anaerobic biodegradability of phthalate esters at 37 degrees C. The observed reduction of PAE biodegradation rates after the thermal pretreatment was found to be proportional to the PAE solubility in water: the higher the solubility, the higher the percentage of the reduction (DEP > DBP > DEHP). PAE were slowly degraded during the pretreatment at 70 degrees C, yet this was probably due to physicochemical reactions than to microbial/biological activity. Therefore, thermal pretreatment of sludge containing PAE should be either avoided or combined with a treatment step focusing on PAE reduction. On the other hand, enzymatic treatment was very efficient in the removal of PAE. The enzymatic degradation of DBP, DEP, and DEHP could be one to two orders of magnitude faster than under normal mesophilic anaerobic conditions. Moreover, the enzymatic treatment resulted in the shortest half-life of DEHP in sludge reported so far. Our study further showed that enzymatic treatment with lipases can be applied to raw sludge and its efficiency does not depend on the solids concentration.     

Isolation and characterization of Sphingomonas sp. GTIN11 capable of carbazole metabolism in petroleum

A bacterial culture was isolated from a manufactured gas plant (MGP) soil based on its ability to metabolize the nitrogen-containing heterocycle carbazole. The culture was identified as a Sphingomonas sp. and was given the designation GTIN11. A cloned 4.2kb DNA fragment was confirmed to contain genes responsible for carbazole degradation. DNA sequence analysis revealed that the fragment contained five open reading frames (ORFs) with the deduced amino acid sequence showing homology to; carbazole terminal dioxygenase (ORF1), 2,3-dihydroxybiphenyl dioxygenase subunits (ORF2 and ORF3), meta-cleavage compound hydrolases (ORF4), and ferrodoxin component of bacterial multicomponent dioxygenases (ORF5). The percent similarity was 61% of these proteins or less to known proteins. The specific activity of Sphingomonas sp. GTIN11 for the degradation of carbazole at 37 degrees C was determined to be 8.0 micromol carbazole degraded/min/g dry cell. This strain is unique in expressing the carbazole degradation trait constitutively. Resting cells of Sphingomonas sp. GTIN11 removed 95% of carbazole and 50% of C1-carbazoles from petroleum in a 16-h treatment time.     

Degradation of diphenyl phthalate by <Emphasis Type="Italic">Sphingomonas chungbukensis</Emphasis>

More than 80% of diphenyl phthalate (DPP) at 100 mg l⁻¹ was degraded by Sphingomonas chungbukensis KCTC 2955 in a mineral salts medium at pH 7.0 and 30°C within 48 h. The maximum specific degradation rate was 5 mg DPP l⁻¹ h⁻¹. It was rapidly converted to monophenyl phthalate and phthalic acid which were further degraded.     

Aerobic degradation of phthalic acid by Comamonas acidovoran Fy-1 and dimethyl phthalate ester by two reconstituted consortia from sewage sludge at high concentrations

Microbial degradation of phthalic acid (PA) and dimethyl phthalate ester (DMPE) under aerobic conditions was investigated using a pure species of bacteria and two consortia from sewage sludge. Five morphologically distinct microorganisms were obtained in pure culture and identified, and tested for the capability of degrading phthalate and DMPE. Comamonas acidovorans strain Fy-1 showed the highest ability to degrade high concentrations of phthalate (2600 mg/l) within 48 h. Two reconstituted consortia of microorganisms, one comprising Pseudomonas fluorescens, P. aureofaciens and Sphingomonas paucimobilis, and the other of Xanthomonas maltophilia and S. paucimobilis, were effective in completely degrading DMPE (400 mg/l) in 48–96 h. The three-species consortium appeared to be more effective in the degradation of DMPE, and both consortia proceeded via formation of mono-methyl phthalate (MMP) and then phthalatic acid before mineralization. This study suggests that high concentrations of the endocrine-disrupting chemicals phthalate and DMPE can be mineralized in wastewater treatment systems by indigenous microorganisms.     

Diethyl phthalate degradation by the freeze-dried,entrapped Bacillus subtilis strain 3C3

Microbial degradation is the key treatment for diethyl phthalate (DEP) of which the efficacy is subdued by substrate toxicity. DEP-degrading Bacillus subtilis strain 3C3 adopted cell size alteration as one of the adaptive mechanisms in response to DEP stress at high concentrations. Nevertheless, to enhance cell tolerance in the protected environment and to facilitate practical treatment operation, cell entrapment was optimized with the entrapment yield at 89 ± 1% in a modified minimal salt medium-containing alginate matrix and the freeze-dried, entrapped cells were then formulated. Among several compounds tested, incorporation of sucrose proved to be beneficial as a cryoprotectant sustaining cell biodegradation efficiency (97%) and viability (≥90%) during freeze drying, storage under a vacuum condition at low temperatures, rehydration and as an additional matrix filler to reinforce the bead structure. The effective DEP treatment of the formulated, entrapped cells was demonstrated in a packed bed continuous system in which 70% DEP removal at hydraulic retention time (HRT) of 30 min was occurred and was enhanced up to 90% when HRT was increased to 60 min. The work demonstrates an effective preparation and a potential application of the formulated entrapped DEP-degrading cells for DEP treatment.     

Sphingomonas astaxanthinifaciens sp. nov., a novel astaxanthin-producing bacterium of the family Sphingomonadaceae isolated from Misasa, Tottori, Japan

A red-pigmented, Gram-negative, motile, strictly aerobic, mesophilic, oval- or short rod-shaped bacterium (TDMA-17(T)) was isolated from fresh water collected at Misasa, a radioactive site in Japan. TDMA-17(T) was slightly tolerant against gamma-ray irradiation, and effectively produced carotenoids (2.8 mg g(-1) dry cells) including, astaxanthin and astaxanthin isomers. Phylogenetic analysis based on 16S rRNA gene sequences placed TDMA-17(T) in a distinct lineage in the family Sphingomonadaceae, and the highest degree of sequence similarity determined were to Sphingomonas aerolata NW12(T) (94.5%), Sphingomonas aurantiaca MA101b(T) (94.0%), Sphingomonas melonis DAPP-PG 224(T) (94.0%), Sphingomonas asaccharolytica IFO 15499(T) (93.9%) and Sphingomonas abaci C42(T) (93.9%). The major fatty acids were C(17 : 1)omega6c (33.0%) and C(18 : 1)omega7c (20.8%). The DNA G+C content was 67.7 mol%. The presence of Q-10 as the main ubiquinone, the presence of Sphingomonadaceae-specific sphingoglycolipid in the polar lipid profiles, the presence of 2-hydroxy fatty acids and the absence of 3-hydroxy fatty acids supported the identification of this strain as a member of the genus Sphingomonas sensu stricto. Phylogenetic distinctiveness and unique phenotypic characteristics differentiated strain TDMA-17(T) from the closely related Sphingomonas species. The results of polyphasic taxonomic analyses suggested that TDMA-17(T) represents a novel Sphingomonas species, for which the name Sphingomonas astaxanthinifaciens sp. nov. is proposed. The type strain is TDMA-17(T) (=NBRC 102146=CCUG 53608).     

Non-specific biodegradation of the organophosphorus pesticides, cadusafos and ethoprophos, by two bacterial isolates

An enrichment culture technique was used for the isolation of microorganisms responsible for the enhanced biodegradation of the nematicide cadusafos in soils from a potato monoculture area in Northern Greece. Mineral salts medium supplemented with nitrogen (MSMN), where cadusafos (10 mg l(-1)) was the sole carbon source, and soil extract medium (SEM) were used for the isolation of cadusafos-degrading bacteria. Two pure bacterial cultures, named CadI and CadII, were isolated and subsequently characterized by sequencing of 16S rRNA genes. Isolate CadI showed 97.4% similarity to the 16S rRNA gene of a Flavobacterium strain, unlike CadII which showed 99.7% similarity to the 16S rRNA gene of a Sphingomonas paucimobilis. Both isolates rapidly metabolized cadusafos in MSMN and SEM within 48 h with concurrent population growth. This is the first report for the isolation and characterization of soil bacteria with the ability to degrade rapidly cadusafos and use it as a carbon source. Degradation of cadusafos by both isolates was accelerated when MSMN was supplemented with glucose. In contrast, addition of succinate in MSMN marginally reduced the degradation of cadusafos. Both isolates were also able to degrade completely ethoprophos, a nematicide chemical analog of cadusafos, but did not degrade the other organophosphorus nematicides tested such as isazofos and isofenphos. Inoculation of a soil freshly treated with cadusafos or ethoprophos (10 mg l(-1)) with high inoculum densities (4.3 x 10(8) cells g(-1)) of Sphingomonas paucimobilis resulted in the rapid degradation of both nematicides. These results indicate the potential of this bacterium to be used in the clean-up of contaminated pesticide waste in the environment.     

BIODEGRADATION OF PHTHALATE ESTERS BY CYANOBACTERIA1

Phthalate esters (PEs) are endocrine‐disrupting pollutants that are ubiquitous in the environment and can be degraded by microorganisms. In this study, we investigated the kinetics and pathway of biodegradation of di‐n‐butyl phthalate (DBP), diethyl phthalate (DEP), and dimethyl phthalate (DMP) by cyanobacteria Anabaena flos‐aquae G. S. West (strain 4054) and two strains of Microcystis aeruginosa (Kütz.) Kütz. (strain 2396 and strain SM). Gas chromatography/mass spectroscopy (GC/MS) and a deuterium‐labeled compound were used to analyze the degrading intermediates. The findings revealed that all three organisms were capable of metabolizing PE, and that among these organisms, A. flos‐aquae achieved the highest degradation. Additionally, the biodegradation of DBP, DEP, and DMP followed first‐order kinetics. Moreover, the results of the enzymatic study suggested that PE was degraded through transesterification on the side chains rather than deesterification. Finally, experiments using deuterium‐labeled DBP showed that there were two degradation pathways: C₁₆→ C₁₄→ C₁₂→ C₁₀→ C₈ and C₁₆→ C₁₅→ C₁₃→ C₁₁→ C₉. Based on our results, the biodegradation pathway of PE for cyanobacteria was suggested.     

Detection and characterization of conjugative degradative plasmids in xenobiotic-degrading Sphingomonas strains

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.     

A direct competitive enzyme-linked immunosorbent assay by antibody coated for diethyl phthalate analysis

A direct competitive enzyme-linked immunosorbent assay (ELISA) has been developed for detection of diethyl phthalate (DEP). Protein-hapten conjugate was synthesized to produce polyclonal antibodies against DEP. Experimental parameters were optimized, including immunoreaction conditions, the dilution ratio of horseradish peroxidase (HRP)-antigen conjugate, time of the antibody coated, effect of pH, and ionic strength. The limit of detection was 0.096 ng/ml, and the linear range was 0.1-3500 ng/ml with a regression coefficient (R²) of 0.9957. Recoveries were between 96.4 and 106.2%. The cross-reactivities of the anti-DEP antibody to six structurally related phthalate esters were less than 9%. The method was successfully applied to the determination of DEP in tap water, river water (Yangtze River), and leachate from plastic drinking bottles. This immunoassay was highly specific, sensitive, rapid, simple, and suitable for DEP monitoring. The results obtained were compared with those obtained using the high-performance liquid chromatography method.     

Anaerobic degradation of phthalic acid esters during digestion of municipal solid waste under landfilling conditions

Anaerobic microorganisms in municipal solid waste samples from laboratory-scale landfill reactors and a pilot-plant biogas digestor were investigated with the aim of assessing their ability to transform four commercially used phthalic acid esters (PAEs) and phthalic acid (PA). The PAEs studied were diethyl phthalate (DEP), butylbenzyl phthalate (BBP), dibutyl phthalate (DBP) and bis(2-ethylhexyl) phthalate (DEHP). No biological transformation of DEHP could be detected in any of the experiments. Together with waste samples from the simulated landfilling conditions, the PAEs (except DEHP) were hydrolytically transformed to their corresponding monoesters. These accumulated as end products, and in most cases they were not further degraded. During incubation with waste from the biogas digestor, the PAEs (except DEHP) were completely degraded to methane and carbon dioxide. The influence of the landfill development phase on the transformations was investigated utilizing PA and DEP as model substances. We found that during both the intense and stable methanogenic (but not the acidogenic) phases, the microoganisms in the samples had the potential to transform PA. A shorter lag phase was observed for the PA transformation in the samples from the stable methanogenic phase as compared with earlier phases. This indicates an increased capacity to degrade PA during the aging phases of the municipal solid waste in landfills. No enhancement of the DEP transformation could be observed as conditions in the methanogenic landfill model changed over a year's time. The results indicate that microorganisms developing in a methanogenic landfill environment have a substantially lower potential to degrade PAEs compared with those developing in a biogas reactor.Abbreviations BBP butylbenzyl phthalate - DEHP bis(2-ethylhexyl) phthalate - CoA coenzyme A - DBP dibutyl phthalate - DEP diethyl phthalate - DS dry solids - MBeP monobenzyl phthalate - MBuP monobutyl phthalate - MEP monoethyl phthalate - MSW municipal solid waste - PA phthalic acid - PAE(s) phthalic acid ester(s) - VFA volatile fatty acids     

Isolation and characterization of a novel sphingomonad capable of growth with chrysene as sole carbon and energy source

A bacterial strain able to grow in pure culture with chrysene as sole added carbon and energy source was isolated from PAH-contaminated soil after successive enrichment cultures in a biphasic growth medium. Initially, growth occurred in the form of a biofilm at the interface between the aqueous and non-aqueous liquid phases. However, after a certain time, a transition occurred in the enrichment cultures, with growth occurring in suspension and a concomitant increase in the rate of chrysene degradation. The strain responsible for chrysene degradation in these cultures, named Sphingomonas sp. CHY-1, was identified by 16S rDNA sequencing as a novel sphingomonad, the closest relative in the databases being Sphingomonas xenophaga BN6T (96% sequence identity). Both these strains clustered with members of the genera Sphingobium and Rhizomonas, but could not be categorically assigned to either genus. Sphingomonas sp. CHY-1 was characterized in terms of its growth on chrysene and other PAH, and the kinetics of chrysene degradation and 14C-chrysene mineralization were measured. At an initial chrysene concentration of 0.5 g l(-1) silicone oil, and an organic/aqueous phase ratio of 1:4, chrysene was 50% degraded after 5 days incubation and 97.5% degraded after 35 days. The protein content of cultures reached a maximum value of 11.5 microg ml(-1) aqueous phase, corresponding to 92 mg g(-1) chrysene. 14C-labelled chrysene was 50% mineralized after 6-8 weeks incubation, 10.7% of the radioactivity was incorporated into cell biomass and 8.4% was found in the aqueous culture supernatant. Sphingomonas sp. CHY-1 also grew on naphthalene, phenanthrene and anthracene, and naphthalene was the preferred substrate, with a doubling time of 6.9 h.