Dominique Patureau

Chemical and biological analysis of endocrine‐disrupting hormones and estrogenic activity in an advanced sewage treatment plant

The steroid hormones estrone (E1), 17β-estradiol (E2), estriol (E3), 17α-ethinylestradiol (EE2), and their conjugated forms were surveyed throughout an advanced sewage treatment plant (STP). The estrogen concentrations in water and sludge samples, collected in October 2004 and April 2005, were determined by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Simultaneously, the estrogenic activity was quantified using estrogen-responsive reporter cell lines (MELN) to investigate the behavior of overall estrogenic compounds. The estrogen concentrations in the inlet ranged from 200 to 500 ng/L, with the contribution of conjugated forms being higher than 50%. The major estrogens in influent were E1 and E3. The estrogenic activity was between 25 and 130 ng/L of E2 equivalents (EEQs). Estrogen concentrations and estrogenicity measured in the inlet and in primary treated sewage were similar, showing a weak impact of primary treatment on hormone removal. In contrast, both estrogen concentration and estrogenicity decreased during biological treatment, with high removal efficiencies (>90%). Estrone, E2, and EE2 persisted in the treated water below 10 ng/L, whereas the estrogenicity was lower than 5 ng/L of EEQs. Estrogen mass flux in the effluent and sludge represented less than 2 and 4%, respectively, of the inlet. Consequently, the fraction of estrogens sorbed into the sludge was very small, and biodegradation was the main vehicle for estrogen elimination. This dual approach, comparing chemical and biological analysis, allowed us to confirm that most of the estrogenic activity occurring in this STP, which receives mainly domestic sewage, resulted from sex hormones.

Aerobic Denitrifiers Isolated from Diverse Natural and Managed Ecosystems

A bstractTwenty-eight bacterial strains were isolated from an ecosystem adapted to fluctuating oxic–anoxic conditions. This ecosystem comprised a mixture of different natural and wastewater treatment environments. Among the 28 strains isolated, 10 exhibited aerobic denitrifying activity, i.e., co-respiration of oxygen and nitrate and simultaneous production of nitrite by 4 of them and of nitrogen gas by the remaining 6. Comparisons between the 16S rDNA sequences of the 10 strains showed that 3 of them were identical to M. aerodenitrificans, whereas RAPD profiles showed that the 3 strains were identical to each other but that they were different from M. aerodenitrificans. This implies that alternating aerobic–anoxic conditions allowed the isolation of a new strain of this aerobic denitrifier. Moreover, other denitrifying bacteria belonging to the genera Paracoccus, Thiobacillus, Enterobacter, Comamonas, and Sphingomonas were isolated in this way. These data imply that a wide variety of bacteria are able to carry out this type of metabolism. M. aerodenitrificans was also detected in methanogenic, denitrifying, nitrifying, phosphate removal, and activated sludge ecosystems by two-step PCR amplification. After 4 months of acclimation to oxic–anoxic phases, the strain was also detected in a canal and in a pond. This suggests that there is no specific natural ecological niche for aerobic denitrifiers but, as soon as selective pressure such as alternating aeration conditions is applied, this metabolism is amplified.

Effect of dissolved oxygen and carbon-nitrogen loads on denitrification by an aerobic consortium.

Abstract Four samples of natural ecosystems and one sample from an activated sludge treatment plant were mixed together and progressively adapted to alternating aerobic/anoxic phases in the presence of nitrate in order to enrich the microflora in aerobic denitrifiers. Aerobic denitrifying performances of this mixed ecosystem at various dissolved oxygen concentrations and various carbon–nitrogen loads were evaluated and compared to those obtained with the aerobic denitrifier Microvirgula aerodenitrificans. The consortium and the pure strain exhibited an aerobic denitrifying activity at air saturation conditions (7 mg dissolved oxygen l–1), i.e. there was co-respiration of the two electron acceptors with significant specific nitrate reduction rates. Dissolved oxygen concentrations had no influence on denitrifying performances above a defined threshold: 0.35 mg l–1 for the consortium and 4.5 mg l–1 for M. aerodenitrificans respectively. Under these thresholds, decreasing the dissolved oxygen concentrations enhanced the denitrifying activity of each culture. The higher the carbon and nitrogen loads, the higher the performance of the aerobic denitrifying ecosystem. However, for M. aerodenitrificans, the nitrate reduction percentage was affected more by variations in nitrogen load than in carbon load.

Microvirgula aerodenitrificans gen. nov., sp. nov., a new Gram-negative bacterium exhibiting co-respiration of oxygen and nitrogen oxides up to oxygen-saturated conditions

A denitrifier micro-organism was isolated from an upflow denitrifying filter inoculated with an activated sludge. The cells were Gram-negative, catalase- and oxidase-positive curved rods and very motile. They were aerobic as well as anoxic heterotrophs that had an atypical respiratory type of metabolism in which oxygen and nitrogen oxides were used simultaneously as terminal electron acceptors. The G&C content was 65 mol%. Our isolate was phenotypically similar to Comamonas testosteroni, according to classical systematic classification systems. However, a phylogenetic analysis based on the 165 rRNA sequence showed that the aerobic denitrifier could not be assigned to any currently recognized genus. For these reasons a new genus and species, Microvirgula aerodenitrificans gen. nov., sp. nov., is proposed, for which SGLY2T is the type strain.

COMBINED PHOSPHATE AND NITROGEN REMOVAL IN A SEQUENCING BATCH REACTOR USING THE AEROBIC DENITRIFIER, MICROVIRGULA AERODENITRIFICANS

A phosphate removal sludge was bioaugmented with the aerobic denitrifier, Microvirgula aerodenitrificans in order to reduce the nitrate produced during the aerobic nitrifying-phosphate uptake phase. Fluorescent in situ hybridization (FISH) was used to follow the fate of the added strain. In order to maintain the pure strain in the complex ecosystem, diverse physiological and kinetic based strategies of bioaugmentation were tested under the sequencing batch reactor (SBR) type culture. The nature of the M. aerodenitrificans inoculum (adapted to nitrate-aerobic conditions or to anoxic one) had no influence on the SBR performances and did not enhance aerobic denitrifying performances. The optimum quantity of the added strain (10% of the total biomass) seemed to have much more positive influence on the long term maintenance of the pure strain than on the SBR performances. A small but daily supply of M. aerodenitrificans gave exactly the same result than a massive and 1-day supply, i.e. no enhancement of performances and no amelioration of the length of maintenance. A continuous supply of carbon during the first hour of the aerobic phase combined to a 10% supply of M. aerodenitrificans gave the best compromise in terms of phosphate removal, nitrification and aerobic denitrification performances. It was accompanied too by a decreased number of the ammonia and nitrite-oxidizing bacteria and a modification of the nitrite-oxidizing floc structure. FISH on M. aerodenitrificans revealed that (i) before bioaugmentation, the strain was already present in the phosphate removal sludge and (ii) the added bacteria almost disappeared from the reactor after 16 HRT. In a last experiment, M. aerodenitrificans embedded in alginate beads allowed enhancement of both aerobic denitrifying performances and length of strain maintenance.

Combined nitrification and denitrification in a single aerated reactor using the aerobic denitrifier Comamonas SP. strain SGLY2

A mixed culture of an aerobic denitrifier, named SGLY2, and autotrophic nitrifiers was carried out either in batch culture or in a single continuous, stirred aerobic reactor. The batch assays were used to show the effect of different carbon sources on the nitrifying activity, and to determine the optimum conditions to maintain the strain SGLY2 in the complex microflora. In particular, conditions of discontinuous addition of the carbon source, as well as nitrifying and denitrifying activities were observed under continuous culture with aerobic reduction of the N-oxides produced by autotrophs into nitrous oxide and dinitrogen.

Occurrence of estrogens in sewage sludge and their fate during plant-scale anaerobic digestion.

Estrogens, which contribute greatly to the endocrine-disrupting activity in sewage, are partially sorbed onto particulate matter during sewage treatment. We thus investigated the occurrence of estrogens in different kinds of sludge and throughout a plant-scale anaerobic digestion process. The analytical method was first validated when sorption interaction between spiked estrogens and sludge could occur. Hence, the recovery ratio of estrone (E1), 17beta-estradiol (E2), estriol (E3) and 17alpha-ethinylestradiol (EE2) were determined when added to liquid sludge and mixed under various conditions. We show that minor non-extractable residues were formed (5-10%), suggesting that the sorption interaction established with sludge did not limit estrogen extraction. Estrogen concentrations measured in collected samples varied with sludge type. Secondary sludge showed higher E1 contents than primary sludge: respectively, 43 and 8 ng g(-1) dry weight (dw). Two pathways of E1 production during secondary treatment are proposed to explain such a result. Higher estrogen concentrations were found in secondary sludge from a conventional plant (55 ng g(-1)dw) compared to those from an advanced plant (13 ng g(-1)dw). Based on estimated estrogen concentrations in sewage, we conclude that operating parameters play a role in the sorption of estrogens during secondary treatment. Also, the hydrophobic properties of the estrogens influenced the individual adsorption of each molecule. Thus, E3 showed the highest estimated concentrations in sewage but very low concentrations in sludge. Finally, plant-scale anaerobic digestion showed low efficiency (<40%) for removing estrogens and, regarding the final dewatering process, concentrations increased for E2 and EE2.

A new dynamic model for bioavailability and cometabolism of micropollutants during anaerobic digestion.

Organic micropollutants (OMPs) are present in wastewater and sludge. Their possible impact to the environment contributes to their increasing scientific and social interest. Anaerobic digestion has been shown as a potential biological process for removal of these compounds. An accurate description of OMP distribution in the environmental system can be used to better understand which compartment is used for degradation and to improve their depletion in conventional wastewater treatment technologies. In this work, we proposed a dynamical model with a four-compartment distribution to describe the Polycyclic Aromatic Hydrocarbons (PAHs) fate during anaerobic digestion. The model is calibrated and validated using experimental data obtained from two continuous reactors fed with primary and secondary sludge operated under mesophilic conditions. A non-linear least square method was used to optimize the model parameters. The resulted model is in accordance with the experimental data. The PAH biodegradation rate is well modeled when considering the aqueous fraction (including free and sorbed to dissolved/colloidal matter PAHs) as the bioavailable compartment. It was also demonstrated in the simulations that the PAHs biodegradation is linked to a mechanism of cometabolism. The model proposed is potentially useful to better understand the micropollutant distribution, predict the fate of PAHs under anaerobic condition and help to optimize the operation process for their depletion.

PAH fate during the anaerobic digestion of contaminated sludge: Do bioavailability and/or cometabolism limit their biodegradation?

The anaerobic removal of 13 Polycyclic Aromatic Hydrocarbons (PAHs) was measured in five continuous anaerobic digestors with different feed sludge, in which abiotic losses were neglected. These feeds were chosen to generate different levels of PAH bioavailability and cometabolism within the reactors. Based on the accurate modelling of PAH sorption in sludge, the aqueous fraction (including free and sorbed-to-dissolved-and-colloidal-matter PAHs) was demonstrated to be bioavailable, which validated a widespread assumption about micropollutants bioavailability in sludge. It was also demonstrated that bioavailability is not the only influencing factor. Indeed, PAHs biodegradation resulted from a combination of bioavailability and cometabolism. An equation adapted from Criddle (1993, The Kinetics of Cometabolism. Biotechnology and Bioengineering 41, 1048-1056) that takes into account both mechanisms was shown to fit the experimental data, with dry matter removal rate identified as the criteria for cometabolism. The existence of a threshold of dry matter cometabolism was suggested, below which PAHs removal would not be possible. The parameters of the Criddle equation were demonstrated to depend on PAH molecular structure, and the results suggest that they would also be influenced by substrate composition and microbial population. This research provided original outcomes for the assessment of micropollutants fate. Indeed, the understanding of the driving mechanisms was improved, which has implications for the optimization of micropollutants removal.

Micropollutant and sludge characterization for modeling sorption equilibria.

The sorption of hydrophobic micropollutants in sludge is one of the major mechanisms which drive their fate within wastewater treatment systems. The objective of this study was to investigate the influence of both sludge and micropollutant characteristics on the equilibria of sorption to particles and to dissolved and colloidal matter (DCM). For this purpose, the equilibrium constants were measured for 13 polycyclic aromatic hydrocarbons, 5 polychlorobiphenyls and the nonylphenol, and five different sludge types encountered in treatment systems: a primary sludge, a secondary sludge, the same secondary sludge after thermal treatment, after anaerobic digestion, and after both treatments. After thermal treatment, no more sorption to DCM was observed. Anaerobic biological treatment was shown to enhance micropollutants sorption to particles and to DCM of one logarithmic unit, due to matter transformation. Partial least-squares linear regressions of sorption data as a function of micropollutant and sludge properties revealed that sludge physical and chemical characteristics were more influential than micropollutant characteristics. Two models were provided to predict the sorption of such micropollutants in any sludge. To our knowledge, this is the first time that a three-compartment approach is used to accurately model micropollutant sorption in sludge and to understand the driving mechanisms.