Identification and isolation of anaerobic, syntrophic phthalate isomer-degrading microbes from methanogenic sludges treating wastewater from terephthalate manufacturing

The microbial populations responsible for anaerobic degradation of phthalate isomers were investigated by enrichment and isolation of those microbes from anaerobic sludge treating wastewater from the manufacturing of terephthalic acid. Primary enrichments were made with each of three phthalate isomers (ortho-, iso-, and terephthalate) as the sole energy source at 37 degrees C with two sources of anaerobic sludge (both had been used to treat wastewater containing high concentrations of phthalate isomers) as the inoculum. Six methanogenic enrichment cultures were obtained which not only degraded the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with concomitant production of methane, presumably involving strictly syntrophic substrate degradation. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant bacteria in the enrichment cultures were affiliated with a recently recognized non-sulfate-reducing subcluster (subcluster Ih) in the group 'Desulfotomaculum lineage I' or a clone cluster (group TA) in the class delta-PROTEOBACTERIA: Several attempts were made to isolate these microbes, resulting in the isolation of a terephthalate-degrading bacterium, designated strain JT, in pure culture. A coculture of the strain with the hydrogenotrophic methanogen Methanospirillum hungatei converted terephthalate to acetate and methane within 3 months of incubation, whereas strain JT could not degrade terephthalate in pure culture. During the degradation of terephthalate, a small amount of benzoate was transiently accumulated as an intermediate, indicative of decarboxylation of terephthalate to benzoate as the initial step of the degradation. 16S rRNA gene sequence analysis revealed that the strain was a member of subcluster Ih of the group 'Desulfotomaculum lineage I', but it was only distantly related to other known species.     

Biodegradation of Phthalate Isomers by Pseudomonas aeruginosa PP4, Pseudomonas sp. PPD and Acinetobacter lwoffii ISP4

Pseudomonas aeruginosa PP4, Pseudomonas sp. PPD and Acinetobacter lwoffii ISP4 capable of utilizing phthalate isomers were isolated from the soil using enrichment culture technique. The strain ISP4 metabolizes isophthalate, while PPD and PP4 utilizes all three phthalate isomers (ortho-, iso- and tere-) as the sole carbon source. ISP4 utilizes isophthalate (0.1%) more rapidly (doubling time, 0.9 h) compared to PPD (4.64 h), PP4 (7.91 h) and other reported strains so far. The metabolic pathways in these isolates were initiated by dihydroxylation of phthalate isomers. Phthalate is hydroxylated to 3,4-dihydro-3,4-dihydroxyphthalate and 4,5-dihydro-4,5-dihydroxyphthalate in strains PP4 and PPD, respectively; while terephthalate is hydroxylated to 2-hydro-1,2-dihydroxyterephthalate. All three strains hydroxylate isophthalate to 4-hydro-3,4-dihydroxyisophthalate. The generated dihydroxyphthalates were subsequently metabolized to 3,4-dihydroxybenzoate (3,4-DHB) which was further metabolized by ortho ring-cleavage pathway. PP4 and PPD cells grown on phthalate, isophthalate or terephthalate showed respiration on respective phthalate isomer and the activity of corresponding ring-hydroxylating dioxygenase, suggesting the carbon source specific induction of three different ring-hydroxylating dioxygenases. We report, for the first time, the activity of isophthalate dioxygenase and its reductase component in the cell-free extracts. The enzyme showed maximum activity with reduced nicotinamide adenine dinucleotide (NADH) in the pH range 8–8.5. Cells grown on glucose failed to respire on phthalate isomers and 3,4-DHB and showed significantly low activities of the enzymes suggesting that the enzymes are inducible.     

Survey of the Anaerobic Biodegradation Potential of Organic Chemicals in Digesting Sludge

The degradation potential of 77 organic chemicals under methanogenic conditions was examined with an anaerobic digesting sludge from the United Kingdom. Degradation was assessed in terms of net total gas (CH₄ plus CO₂) produced, expressed as a percentage of the theoretical production (ThGP). The compounds tested were selected from various chemical groups and included substituted phenols and benzoates, pesticides, phthalic acid esters, homocyclic and heterocyclic ring compounds, glycols, and monosubstituted benzenes. The results obtained were in good agreement with published surveys of biodegradability in U.S. digesting sludges and other methanogenic environments. In general, the presence of chloro or nitro groups inhibited anaerobic gas production, while carboxyl and hydroxyl groups facilitated biodegradation. The relationship between substituent position and susceptibility to methanogenic degradation was compound dependent. The following chemicals were completely degraded (≥80% ThGP) at a concentration of 50 mg of carbon per liter: phenol, 2-aminophenol, 4-cresol, catechol, sodium benzoate, 4-aminobenzoic acid, 3-chlorobenzoic acid, phthalic acid, ethylene glycol, diethylene glycol, triethylene glycol, sodium stearate, and quinoline. 3-Cresol, 4-chlorobenzoic acid, dimethyl phthalate, and pyridine were partially degraded. Although the remaining chemicals tested were either persistent or toxic, their behavior may differ at more environmentally realistic chemical-to-biomass ratios. Our findings suggest that biodegradability assessments made with sludge from one source can be extrapolated to sludge from another source with a reasonable degree of confidence and should help in predicting the fate of an organic chemical during the anaerobic digestion of sewage sludge.     

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.     

Biodegradation of phthalic acid esters in river water and activated sludge.

The primary and ultimate biodegradability of phthalic acid, monobutyl phthalate, and five structurally diverse phthalic acid ester plasticizers in river water and activated sludge samples were determined via ultraviolet spectrophotometry, gas chromatography, and CO2 evolution. The compounds studied underwent rapid primary biodegradation in both unacclimated river water and acclimated activated sludge. When activated sludge acclimated to phthalic acid esters was used as the inoculum for the CO2 evolution procedure, greater than 85% of the total theoretical CO2 was evolved. These studies demonstrate that the phthalic acid ester plasticizers and intermediate degradation products readily undergo ultimate degradation in different mixed microbial systems at concentrations ranging from 1 to 83 mg/liter.     

Degradation of Dimethyl Isophthalate by Viarovorax paradoxus Strain T4 Isolated from Deep-Ocean Sediment of the South China Sea

Viarovorax paradoxusT4 strain was isolated from deep-ocean sediment and demonstrated to be able to degrade dimethyl isophthalate (DMI). When DMI was utilized as the sole source of carbon and energy, it was transformed by hydrolysis initially, forming monomethyl isophthalate (MMI) and isophthalate acid (IA) as degradation intermediates. DMI and MMI were completely transformed to MMI and IA in about 100 h, respectively. Degradation of IA was completed in about 55 h. Analysis of total organic carbon in the culture medium confirmed that more than 80% of the substrate carbon was mineralized. Bacterial esterase induced by a range of substrates could be assessed using p-nitrophenyl acetate as the common substrate using crude enzyme preparation. The decreasing trend of K _m values derived from the Michaelis–Menten equation was dimethyl phthalate (DMP) > monomethyl phthalate (MMP) > dimethyl terephthalate (DMT) > Liver esterase > DMI > MMI > monomethyl terephthalate (MMT), indicating that higher K _m values were obtained by di-esters than mono-ester and the esters induced by terephthalate esters showed the highest activity. This investigation suggests that biochemical pathways for phthalate esters share many common characteristics and the esterases induced by different substrates are highly specific.     

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.     

Anaerobic degradation of xenobiotics by organisms from municipal solid waste under landfilling conditions

The potential for biological transformation of 23 xenobiotic compounds by microorganisms in municipal solid waste (MSW) samples from a laboratory scale landfill reactor was studied. In addition the influence of these xenobiotic compounds on methanogenesis was investigated. All R11, 1,1 dichloroethylene, 2,4,6 trichlorophenol, dimethyl phthalate, phenol, benzoate and phthalic acid added were completely transformed during the period of incubation (> 100 days). Parts of the initially added perchloroethylene, trichloroethylene, R12, R114, diethyl phthalate, dibutyl phthalate and benzylbutyl phthalate were transformed. Methanogenesis from acetate was completely inhibited in the presence of 2,5 dichlorophenol, whereas 2,4,6 trichlorophenol and R11 showed an initial inhibition, whenafter methane formation recovered. No transformation or effect on the anaerobic microflora occurred for R13, R22, R114, 3 chlorobenzoate, 2,4,6 trichlorobenzoate, bis(2 ethyl)hexyl phthalate, diisodecyl phthalate and dinonyl phthalate. The results indicate a limited potential for degradation, of the compounds tested, by microorganisms developing in a methanogenic landfill environment as compared with other anaerobic habitats such as sewage digestor sludge and sediments.Abbreviations BBP benzylbutylphthalate - DEHP bis(2 ethylhexyl) phthalate - 3 CB 3 chlorobenzoate - R22 chlorodifluoromethane - CFC chlorofluorocarbon - R13 chlorotrifluoromethane - cis1,2 DCE cis 1,2 dichloroethylene - DBP dibutyl phthalate - R12 dichlorodifluoromethane - 1,1 DCE 1,1 dichloroethylenel - R114 dichlorotetrafluoroethane - 2,5 DCP 2,5 dichlorophenol - DEP diethyl phthalate - DiDP diisodecyl phthalate - DMP Dimethyl phthalate - DNP dinonyl phthalate - MSW dunicipal solid waste - PCE perchloroethylene - PA phthalic acid - PAE phthalic acid esters - R11 trichlorofluoromethane - 2,4,6 TCB 2,4,6 trichlorobenzoate - 2,4,6 TCP 2,4,6 trichlorophenol - VC vinylchloride     

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.     

Microbial characterization and quantification of an anaerobic sludge degrading dimethyl phthalate

Aims:  Characterization and quantification of microbial community in dimethyl phthalate (DMP)-degrading anaerobic sludge using molecular techniques.
Methods and Results:  An enriched anaerobic sludge effectively degrading over 99% of dimethyl phthalate in an upflow anaerobic sludge blanket (UASB) reactor for 530 days was characterized and quantified by 16S rRNA-based molecular methods. A total of 78 Bacteria clones were classified into 22 operational taxonomic units (OTUs) in nine divisions, including Firmicutes , Proteobacteria, Chloroflexi, Thermotogae , Bacteroidetes/Chlorobi , Spirochaetes , Acidobacteria and two candidate divisions. The two most abundant OTUs were likely responsible, respectively, for the de-esterification of DMP and the subsequent phthalate degradation. The outer layer of the granule was dominated by Bacteria; whereas the interior was by Archaea , of which 89 ± 5% were acetoclastic Methanosaetaceae and 11 ± 5% hydrogenotrophic Methanomicrobiales .
Conclusions:  Twenty-two Bacteria OTUs in DMP-degrading anaerobic sludge distributed in nine divisions. The two most abundant OTUs were likely responsible respectively for the de-esterification of DMP and the subsequent phthalate degradation. Layered granular microstructure of DMP-degrading anaerobic sludge suggested that the rate of DMP de-esterification is faster than its inward diffusion rate.
Significance and Impact of the Study:  This work is the first study to characterize and quantify the microbial community in the anaerobic phthalic ester degrading sludge from the anaerobic reactor.     

Activated Sludge Biodegradation of 12 Commercial Phthalate Esters

The activated sludge biodegradability of 12 commercial phthalate esters was evaluated in two test systems: (i) a semicontinuous activated sludge test and (ii) an acclimated 19-day die-away procedure. Both procedures demonstrated that phthalate esters are rapidly biodegraded under activated sludge conditions when loss of the parent phthalate ester (primary degradation) is measured.     

Degradation of Phthalic Acids by Denitrifying, Mixed Cultures of Bacteria

Mixed cultures of bacteria, enriched from aquatic sediments, grew anaerobically on all three isomers of phthalic acid. Each culture grew anaerobically on only one isomer and also grew aerobically on the same isomer. Pure cultures were isolated from the phthalic acid (o-phthalic acid) and isophthalic acid (m-phthalic acid) enrichments that grew aerobically on phthalic and isophthalic acids. Cell suspension experiments indicated that protocatechuate is an intermediate of aerobic catabolism. Pure cultures which grew aerobically on terephthalic acid (p-phthalic acid) could not be isolated from the enrichments, and neither could pure cultures that grew anaerobically on any of the isomers. Cell suspension experiments suggested that separate pathways exist for the aerobic and anaerobic oxidation of phthalic acids. Each enrichment culture used only one phthalic acid isomer under anaerobic conditions, but all isomers were simultaneously adapted for the anaerobic catabolism of benzoate. Cells grown anaerobically on a phthalic acid immediately attacked the isomer under anaerobic conditions, whereas there was a lag before aerobic breakdown occurred, and, for phthalic and terephthalic acids, chloramphenicol stopped aerobic adaptation but had no effect on anaerobic catabolism. This work suggests that phthalic acids are biodegradable in anaerobic environments.     

Biodegradation of di-n-butyl phthalate by Rhodococcus sp. JDC-11 and molecular detection of 3, 4-phthalate dioxygenase gene

Rhodococcus sp. JDC-11, capable of utilizing di-n-butyl phthalate (DBP) as the sole source of carbon and energy, was isolated from sewage sludge and confirmed mainly based on 16S rRNA gene sequence analysis. The optimum pH, temperature, and agitation rate for DBP degradation by Rhodococcus sp. JDC-11 was 8.0, 30 degrees C, and 175 rpm, respectively. In addition, the effect of glucose concentration on DBP degradation indicated that low concentration of glucose inhibited the degradation of DBP while high concentrations of glucose increased its degradation. Meanwhile, the substrates utilization test showed that JDC-11 could also utilize other phthalates. Furthermore, the major metabolites of DBP degradation were identified as mono-butyl phthalate and phthalic acid by gas chromatography-mass spectrometry and the metabolic pathway of DBP degradation by Rhodococcus sp. JDC-11 was tentatively speculated. Using a set of new degenerate primer, partial sequence of the 3, 4-phthalate dioxygenase gene was obtained from the strain. Sequence analysis revealed that the phthalate dioxygenase gene of JDC-11 was highly homologous to the large subunit of phthalate dioxygenase from Rhodococcus coprophilus strain G9.     

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     

Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction

We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.     

Bacterial degradation of phthalate isomers and their esters

Phthalate isomers and their esters are used heavily in various industries. Excess use and leaching from the product pose them as major pollutants. These chemicals are toxic, teratogenic, mutagenic and carcinogenic in nature. Various aspects like toxicity, diversity in the aerobic bacterial degradation, enzymes and genetic organization of the metabolic pathways from various bacterial strains are reviewed here. Degradation of these esters proceeds by the action of esterases to form phthalate isomers, which are converted to dihydroxylated intermediates by specific and inducible phthalate isomer dioxygenases. Metabolic pathways of phthalate isomers converge at 3,4-dihydroxybenzoic acid, which undergoes either ortho- or meta- ring cleavage and subsequently metabolized to the central carbon pathway intermediates. The genes involved in the degradation are arranged in operons present either on plasmid or chromosome or both, and induced by specific phthalate isomer. Understanding metabolic pathways, diversity and their genetic regulation may help in constructing bacterial strains through genetic engineering approach for effective bioremediation and environmental clean up.     

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.     

Increased biogas production at wastewater treatment plants through co-digestion of sewage sludge with grease trap sludge from a meat processing plant

The feasibility of co-digesting grease trap sludge from a meat-processing plant and sewage sludge was studied in batch and reactor experiments at 35 degrees C. Grease trap sludge had high methane production potential (918 m(3)/tVS(added)), but methane production started slowly. When mixed with sewage sludge, methane production started immediately and the potential increased with increasing grease trap sludge content. Semi-continuous co-digestion of the two materials was found feasible up to grease trap sludge addition of 46% of feed volatile solids (hydraulic retention time 16d; maximum organic loading rate 3.46 kgVS/m(3)d). Methane production was significantly higher and no effect on the characteristics of the digested material was noticed as compared to digesting sewage sludge alone. At higher grease trap sludge additions (55% and 71% of feed volatile solids), degradation was not complete and methane production either remained the same or decreased.     

Transformation of indole by methanogenic and sulfate-reducing microorganisms isolated from digested sludge

In the present study, mineralization of an aromaticN-heterocyclic molecule, indole, by microorganisms present in anaerobically digested sewage sludge was examined. The first step in indole mineralization was the formation of a hydroxylated intermediate, oxindole. The rate of transformation of indole to oxindole and its subsequent disappearance was dependent on the concentration of inoculum and indole and the incubation temperature. Methanogenesis appeared to be the dominant process in the mineralization of indole in 10% digested sludge even in the presence of high concentrations of sulfate. Enrichment of the digested sludge with sulfate as an electron acceptor allowed the isolation of a metabolically stable mixed culture of anaerobic bacteria which transformed indole to oxindole and acetate, and ultimately to methane and carbon dioxide. This mixed culture exhibited a predominance of sulfate-reducers over methanogens with more than 75% of the substrate mineralized to carbon dioxide. The investigation demonstrates that indole can be transformed by both methanogenic and sulfate-reducing microbial populations.