Removal of Phthalate Esters in Soil Column Inoculated with Microorganisms

The removal of phthalate esters, such as di-2-ethyl hexyl phthalate (DEHP) was efficiently effected by inoculating and retaining viable cells of Nocardia erythropolis, a bacterium known capable of rapidly degrading phthalate esters, in soil column. When an influent containing 3000 ppm of DEHP was passed through a column inoculated with Nocardia erythropolis, the eluent from the column was gas-chromatographically free of DEHP after 1 day. Residual DEHP on the support after 32 days in the column inoculated with Nocardia erythropolis was only 0.14% against the total amount of DEHP fed, whereas it was 5.2% in the uninoculated column. Microorganisms capable of utilizing DEHP were isolated from the inoculated and un- inoculated columns after 32 days operation and identified. The DEHP utilizing microorganisms in the inoculated column were found to belong to Nocardia erythropolis, Nocardia restricta and Pseudomonas putida (Biotype B), and those in the uninoculated column to Nocardia erythropolis, Pseudomonas putida (Biotype A and B) and Pseudomonas acidovorans. In particular, strain 1-1 of Nocardia erythropolis isolated from the inoculated column was morphologically and biochemically identical with the inoculated Nocardia erythropolis S-l. Ratio of all Nocardia erythropolis to total cells recovered increased from 10.8% immediately after inoculation to 27.2% after 32 days in inoculated column.     

Effect of Rifampicin and Uracil Depletion on Bacteriophage ϕX174 DNA Synthesis in an E. coli dnaHts Mutant

The fluorescent antibody technique was used to trace an inoculated Nocardia erythropolis strain which was capable of rapidly degrading phthalate esters in soil column and activated sludge systems. The reaction of antibody to Nocardia erythropolis S-1 was highly strain specific, i. e., only one of twelve other strain of N. erythropolis was stained with this fluorescent antibody. All other species of Nocardia and other genera of bacteria and a strain of Candida were not stained. Using this technique it was demonstrated that N. erythropolis S-1 inoculated into activated sludge and soil column systems was successfully distinguished from many other microorganisms in mixed culture systems, and the distribution of this strain was appreciated.     

Purification and some properties of a phthalate ester hydrolyzing enzyme from Nocardia erythropolis

Summary A phthalate ester hydrolyzing enzyme has been purified from the culture broth of Nocardia erythropolis, a Gram-positive bacterium capable of degrading phthalate esters rapidly. The purified enzyme appeared homogeneous on polyacrylamide gel disc-electrophoresis, and its molecular weight was estimated to be about 15,000. The optimal pH and temperature were pH 8.6 and 42°C, respectively. The enzyme was stable in a pH range from 7.0 to 8.0 and below 30°C. The enzyme activity was stimulated by Ca²⁺ and taurocholate, but inhibited by several metals such as Hg²⁺. Most of the phthalate esters tested were hydrolyzed to phthalate and alcohols regardless of the type of side-chain. In addition, the enzyme rapidly hydrolyzed olive oil and tributyrin. This enzyme from N. erythropolis may be a novel type of lipase with broad substrate specificity.Microbial degradation of phthalate esters. Part X     

Identification of Phthalate Ester-assimilating Bacteria

Seventeen bacterial capable of utilizing phthalate esters isolated from natural sources were identified. Based on morphological and biochemical characteristics, type of cell division, GC content in DNA, principal amino acid in the cell wall and cellular fatty acid composition, 10 isolates were identified as Nocardia erythropolis, one isolate as Pseudomonas acidovorans, another as Pseudomonas cepacia and four as members of the genus of Corynebacterium.     

Degradation of environmental endocrine disruptor di-2-ethylhexyl phthalate by a newly discovered bacterium, Microbacterium sp. strain CQ0110Y

In this study di-2-ethylhexyl phthalate (DEHP)-degradation strain CQ0110Y was isolated from activated sludge. According to the biophysical/biochemical characteristics and analysis of 16S rDNA, the strain was identified as Microbacterium sp. The results of this study showed the optimal pH value and optimal temperature which influenced the degradation rate in wastewater: pH 6.5–7.5, 25–35°C. Kinetics of degradation reaction had been performed at different initial concentrations and different time. Analyzed with SPSS10.0 software, the DEHP degradation can be described as the same exponential model when the initial DEHP concentration was lower than 1,350 mg/l. The kinetics equation was ln C = −0.4087t + A, with the degradation half life of DEHP in wastewater (1.59 days). To the best of our knowledge, this is the first reported case of DEHP degradation by Microbacterium sp. strain. Xiang Li and Ji-an Chen contributed equally to this work.     

The effects of hydraulic retention time and sludge retention time on the fate of di-(2-ethylhexyl) phthalate in a laboratory-scale anaerobic-anoxic-aerobic activated sludge system

A laboratory-scale anaerobic-anoxic-aerobic (AAA) activated sludge wastewater treatment system was employed to investigate the effects of hydraulic retention time (HRT) and sludge retention time (SRT) on the removal and fate of di-(2-ethylhexyl) phthalate (DEHP). In the range from 5 to 14h, HRT had no significant effect on DEHP removal. However, longer HRT increased DEHP accumulation in the system and DEHP retention in the waste sludge. When SRT was increased from 15 to 25d, DEHP removal efficiency stayed above 96%. Compared to the removal of only 88% at SRT of 10d, longer SRT enhanced DEHP degradation efficiency. The optimal HRT and SRT for both nutrients (nitrogen and phosphorus) and DEHP removal were 8h and 15d. At these retention times, about 71% of DEHP was degraded by the activated sludge process, 26% was accumulated in the system, 2% was released in the effluent, and 1% remained in the waste sludge. The anaerobic, anoxic and aerobic reactors were responsible for 15%, 19% and 62% of the overall DEHP removal, respectively.     

Isolation of Microorganisms Growing on Phthalate Esters and Degradation of Phthalate Esters by Pseudomonas acidovorans 256–1

Many microorganisms (17 strains of gram-positive bacteria, 8 strains of gram-negative bacteria, 2 strains of fungi) capable of assimilating di-2-ethyl hexyl phthalate (DEHP) were isolated from soil and other natural sources. When Pseudomonas acidovorans 256–1 among these microorganisms was aerobically cultured in media containing 0.5 % of DEHP, DEHP disappeared completely in 72 hr when assayed gaschromatographically. Most of phthalate esters could be assimilated, regardless of their side-chain types. In addition, branched-alkyl phthalate was assimilated better than n-alkyl phthalate. Based on degradation rate of n-alkyl phthalate in relation to its side chain and carbon number, two peaks were observed in n-alkyl phthalate with four and seven carbon number on its side-chain.     

Efficiency evaluation of activated sludge treatments of wastewater from fiber board manufacturing

Summary Wastewater from fiber board manufacture consisting in a mixture of Pinus radiata, Eucaliptus globulus and Laureliopsis phillipiana (tepa) (3:1:1) has been studied in laboratory scale activated sludge reactors with organic load rate range of 50–1700 gCOD/m³.d. A stable operation at high organic load rate with hydraulic retention time of one day was achieved. Purification efficiencies up to 90 % of COD removal could be achieved in an activated sludge treatment of fiber board wastewater working with 1 day HRT for wood log cooking wastewater and with 4 days HRT when glueing wastewater is added to the cooking wastewater treatment. Suspended solids, color and phenol concentration were negligible in the efluent of the activated sludge system.     

Biodegradation of sulfamethoxazole by individual and mixed bacteria

Antibiotic compounds, like sulfamethoxazole (SMX), have become a concern in the aquatic environment due to the potential development of antibacterial resistances. Due to excretion and disposal, SMX has been frequently detected in wastewaters and surface waters. SMX removal in conventional wastewater treatment plants (WWTPs) ranges from 0% to 90%, and there are opposing results regarding its biodegradability at lab scale. The objective of this research was to determine the ability of pure cultures of individual and mixed consortia of bacteria (Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas putida, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus rhodocrous, and Rhodococcus zopfii) known to exist in WWTP activated sludge to remove SMX. Results showed that R. equi alone had the greatest ability to remove SMX leading to 29% removal (with glucose) and the formation of a metabolite. Degradation pathways and metabolite structures have been proposed based on the potential enzymes produced by R. equi. When R. equi was mixed with other microorganisms, a positive synergistic effect was not observed and the maximum SMX removal achieved was 5%. This indicates that pure culture results cannot be extrapolated to mixed culture conditions, and the methodology developed here to study the biodegradability of compounds under controlled mixed culture conditions offers an alternative to conventional studies using pure bacterial cultures or inocula from activated sludge sources consisting of unknown and variable microbial populations.     

Heat shock protein synthesis is induced by diethyl phthalate but not by di(2-ethylhexyl) phthalate in radish (Raphanus sativus)

The toxicity and effects on protein synthesis of the phthalate esters diethyl phthalate (DEP) and di(2-ethylhexyl) phthalate (DEHP) was studied in radish seedlings (Raphanus sativus cv. Kööpenhaminan tori). Phthalate esters are a class of commercially important compounds used mainly as plasticizers in high molecular-weight polymers such as many plastics. They can enter soil through various routes and can affect plant growth and development. First the effect of DEP and DEHP on the growth of radish seedlings was determined in an aqueous medium. It was found that DEP, but not DEHP, caused retardation of growth in radish. A further investigation on protein synthesis during DEP-stress was executed by in vivo protein labeling combined with two-dimensional gel electrophoresis (2D-PAGE). For comparisons with known stress-induced proteins a similar experiment was done with heat shock, and the induced heat shock proteins (HSPs) were compared with those of DEP-stress. The results showed that certain HSPs can be used as an indicator of DEP-stress, although the synthesis of most HSPs was not affected by DEP. DEP also elicited the synthesis of numerous proteins found only in DEP-treated roots. The toxic effect of phthalate esters and the roles of the induced proteins are discussed.     

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.     

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.     

Integrated biosorption and photocatalytic oxidation treatment of di(2-ethylhexyl)phthalate

Di(2-ethylhexyl)phthalate (DEHP), a toxic phthalate ester, is a ubiquitous contaminant due to its extensive use and persistence. No available treatment method can cost-effectively remove it from industrial wastewater. In a previous study, DEHP was effectively removed from aqueous solution by adsorption onto the biomass of selected seaweed, i.e., beached seaweed and Sargassum siliquastrum. Since biosorption cannot detoxify DEHP, the degradation (and detoxification) of desorbed DEHP from seaweed biomass by photocatalytic oxidation (PCO) was attempted. The first part of this study was to optimize the conditions for the degradation of desorbed DEHP in aqueous solution by PCO. Under optimized conditions, a total degradation of 20 mg/L of DEHP was achieved within 45 min. The degradation intermediates/products such as phthalic anhydride and 2-ethyhexanol were identified by GC-MS analysis. Total organic carbon analysis was also used to ensure the complete mineralization of DEHP. The Microtox^® test was used to assess the toxicities of the parent and degraded compounds. In the second part of this study, DEHP was first removed and concentrated by adsorption onto seaweed biomass under the conditions optimized in the previous study. It was then desorbed from seaweed biomass and degraded by PCO. Results indicate that the treatment for DEHP by integrating biosorption and PCO is feasible.     

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.     

Use of claydite-immobilized oil-oxidizing microbial cells for purification of water from oil

Oil-oxidizing bacteria were isolated from oil-polluted soil and water samples and identified as Acinetobacter calcoaceticus K-4, Nocardia vaceinii K-8, Rhodococcus erythropolis EK-1, and Mycobacterium sp. K-2. It was found that immobilization of the bacteria on an expanded clay aggregate accelerated their growth and consumption of hydrocarbon substrates. It was also found that water polluted with 100 mg/l oil could be purified with Rhodococcus erythropolis EK-1 and Nocardia vaceinii K-8 cells immobilized in this way. The dependence of the degree of water purification on its flow rate, aeration, and availability of nitrogen and phosphorus sources was determined. The efficiency of water purification from oil by immobilized Rhodococcus erythropolis EK-1 cells at high flow rates (of up to 0.68 l/h), low aeration (of 0.1 l/l per min) and an intermittent supply of 0.01% diammonium phosphate reached 99.5–99.8%.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 58–63.Original Russian Text Copyright © 2005 by Pirog, Shevchuk, Voloshina, Gregirchak.     

Biodegradation of coal slurry transport wastewater organics

Summary Activated sludge was successful in reducing the levels of dissolved organic carbon (DOC) in coal slurry wastewaters. DOC removal by the activated sludge ranged from 61% to 97% with a large percentage (21–41%) of this DOC being completely metabolized to CO₂. Second order kinetic constants (k ₂) developed for DOC removal ranged from 1.39·10^–4 to 2.30·10^–1 liter·day^–1·(mg of sludge)^–1, providing evidence that biological treatment was an effective mechanism for reducing the pollution potential of the slurry wastewaters. After treatment with activated sludge a residual DOC remained in the wastewater and data from ultrafiltration studies indicated that this residual carbon was of MW>1000. The activated sludge preferentially removed the lower (MW<1000) molecular weight compounds and the higher molecular weight DOC was more resistant to biological attack. However, extended acclimation (greater than 1 month) enabled the activated sludge to remove the higher molecular weight DOC from the slurry wastewaters.     

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     

Degradation of Phthalate and Di-(2-Ethylhexyl)phthalate by Indigenous and Inoculated Microorganisms in Sludge-Amended Soil

The metabolism of phthalic acid (PA) and di-(2-ethylhexyl)phthalate (DEHP) in sludge-amended agricultural soil was studied with radiotracer techniques. The initial rates of metabolism of PA and DEHP (4.1 nmol/g [dry weight]) were estimated to be 731.8 and 25.6 pmol/g (dry weight) per day, respectively. Indigenous microorganisms assimilated 28 and 17% of the carbon in [¹⁴C]PA and [¹⁴C]DEHP, respectively, into microbial biomass. The rates of DEHP metabolism were much greater in sludge assays without soil than in assays with sludge-amended soil. Mineralization of [¹⁴C]DEHP to ¹⁴CO₂ increased fourfold after inoculation of sludge and soil samples with DEHP-degrading strain SDE 2. The elevated mineralization potential was maintained for more than 27 days. Experiments performed with strain SDE 2 suggested that the bioavailability and mineralization of DEHP decreased substantially in the presence of soil and sludge components. The microorganisms metabolizing PA and DEHP in sludge and sludge-amended soil were characterized by substrate-specific radiolabelling, followed by analysis of ¹⁴C-labelled phospholipid ester-linked fatty acids (¹⁴C-PLFAs). This assay provided a radioactive fingerprint of the organisms actively metabolizing [¹⁴C]PA and [¹⁴C]DEHP. The ¹⁴C-PLFA fingerprints showed that organisms with different PLFA compositions metabolized PA and DEHP in sludge-amended soil. In contrast, microorganisms with comparable ¹⁴C-PLFA fingerprints were found to dominate DEHP metabolism in sludge and sludge-amended soil. Our results suggested that indigenous sludge microorganisms dominated DEHP degradation in sludge-amended soil. Mineralization of DEHP and PA followed complex kinetics that could not be described by simple first-order equations. The initial mineralization activity was described by an exponential function; this was followed by a second phase that was described best by a fractional power function. In the initial phase, the half times for PA and DEHP in sludge-amended soil were 2 and 58 days, respectively. In the late phase of incubation, the apparent half times for PA and DEHP increased to 15 and 147 days, respectively. In the second phase (after more than 28 days), the half time for DEHP was 2.9 times longer in sludge-amended soil assays than in sludge assays without soil. Experiments with radiolabelled DEHP degraders suggested that a significant fraction of the ¹⁴CO₂ produced in long-term degradation assays may have originated from turnover of labelled microbial biomass rather than mineralization of [¹⁴C]PA or [¹⁴C]DEHP. It was estimated that a significant amount of DEHP with poor biodegradability and extractability remains in sludge-amended soil for extended periods of time despite the presence of microorganisms capable of degrading the compound (e.g., more than 40% of the DEHP added is not mineralized after 1 year).     

Effects of three phthalate esters on the life-table demography of freshwater rotifer <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis> Pallas

Phthalate esters (PAEs) are mainly used in the polymer industry as external plasticizers in PVC, and tend to migrate slowly out of the plastic, either into the air by volatilization or into water or other solvents by dissolution. Di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP) and di-2-ethylhexyl phthalate (DEHP) are three members of PAEs, identified as priority controlled hazardous substances by the United States Environmental Protection Agency, and have been shown to have potential for endocrine disrupting effects on vertebrates and humans. The effects of DBP, BBP and DEHP on survival and reproduction of the freshwater rotifer Brachionus calyciflorus were studied using life-table demographic methods. The results showed that all the life-table demographic parameters of B. calyciflorus were markedly affected by DBP and BBP, but not by DEHP. The net reproductive rate representing the output of reproduction was more affected than all the other parameters representing population growth, development or survival of the rotifers. Compared to the solvent control, DBP and BBP, both at 500 μg l⁻¹, significantly increased the net reproductive rate, and prolonged the generation time and the life expectancy at hatching of the rotifers. DBP at 50 μg l⁻¹ markedly decreased the intrinsic rate of population increase of the rotifers, but the reverse was true for BBP at 50 and 500 μg l⁻¹. Among all the parameters, the intrinsic rate of population increase was the most sensitive to DBP and BBP. The levels of PAEs in water from all the studied rivers and lakes in the world did not affect the population growth of rotifers.     

Fungal biodegradation of phthalate plasticizer in situ

This unique study describes how Aspergillus japonicus, Penicillium brocae and Purpureocillium lilacinum, three novel isolates of our laboratory from heavily plastics-contaminated soil completely utilized the plasticizer di(2-ethylhexyl)phthalate (DEHP) bound to PVC blood storage bags (BB) in simple basal salt medium (BSM) by static submerged growth (28 °C). Initial quantification as well as percentage utilization of DEHP blended to BB were estimated periodically by extracting it into n-hexane. A two-stage cultivation strategy was employed for the complete mycoremediation of DEHP from BB in situ. During the first growth stage, about two-third parts of total (33.5 % w/w) DEHP bound to BB were utilized in two weeks, accompanied by increased fungal biomass (~0.15–0.32 g per g BB) and sharp declining (to ~3) of initial pH (7.2). At this stagnant growth state (low pH), spent medium was replaced by fresh BSM (pH, 7.2), and thus in the second stage the remaining DEHP (one-third) in BB was utilized completely. The ditches and furrows seen from the topology of the BB as seen by the 3D AFM image further confirmed the bioremediation of DEHP physically bound to BB in situ. Of the three mycelial fungi employed, P. lilacinum independently showed highest efficiency for the complete utilization of DEHP bound to BB, whose activity was comparable to that of the consortium comprising all the three fungi described herein. To sum up, the two-stage cultivation strategy demonstrated in this study shows that a batch process would efficiently remediate the phthalic acid esters blended in plastics on a large scale, and thus it offers potentials for the management of plastics wastes.