Biodegradation of estrogens by facultative anaerobic iron-reducing bacteria

Natural estrogens such as estrone, 17β-estradiol, estriol, and the synthetic component of contraceptive pills, 17α-ethinylestradiol, enter the municipal wastewater treatment plant via human excretions. A significant portion of these substances is found to remain in reject water produced after anaerobic digestion of activated sludge. In this study, the effect of the oxidant, Fe(III), and facultative anaerobic strain of iron-reducing bacteria on the anaerobic degradation of estrogens in reject water was investigated. Synthetic 17α-ethinylestradiol remained resistant to anaerobic biodegradation by iron-reducing bacteria, while natural estrogens such as 17β-estradiol, estriol, and estrone were removed by 92%, 60% and 27%, respectively, after 15 days of batch cultivation of iron-reducing bacteria in reject water with the addition of all estrogens to concentrations 100 μg l⁻¹ each. The ability of facultative anaerobic iron-reducing bacteria to degrade estrogens can be used for the anaerobic removal of trace organics from reject water in municipal wastewater treatment plant.     

Effect of concentration and hydraulic reaction time on the removal of pharmaceutical compounds in a membrane bioreactor inoculated with activated sludge

Pharmaceuticals are often not fully removed in wastewater treatment plants (WWTPs) and are thus being detected at trace levels in water bodies all over the world posing a risk to numerous organisms. These organic micropollutants (OMPs) reach WWTPs at concentrations sometimes too low to serve as growth substrate for microorganisms; thus, co-metabolism is thought to be the main conversion mechanism. In this study, the microbial removal of six pharmaceuticals was investigated in a membrane bioreactor at increasing concentrations (4–800 nM) of the compounds and using three different hydraulic retention times (HRT; 1, 3.5 and 5 days). The bioreactor was inoculated with activated sludge from a municipal WWTP and fed with ammonium, acetate and methanol as main growth substrates to mimic co-metabolism. Each pharmaceutical had a different average removal efficiency: acetaminophen (100%) > fluoxetine (50%) > metoprolol (25%) > diclofenac (20%) > metformin (15%) > carbamazepine (10%). Higher pharmaceutical influent concentrations proportionally increased the removal rate of each compound, but surprisingly not the removal percentage. Furthermore, only metformin removal improved to 80–100% when HRT or biomass concentration was increased. Microbial community changes were followed with 16S rRNA gene amplicon sequencing in response to the increment of pharmaceutical concentration: Nitrospirae and Planctomycetes 16S rRNA relative gene abundance decreased, whereas Acidobacteria and Bacteroidetes increased. Remarkably, the Dokdonella genus, previously implicated in acetaminophen metabolism, showed a 30-fold increase in abundance at the highest concentration of pharmaceuticals applied. Taken together, these results suggest that the incomplete removal of most pharmaceutical compounds in WWTPs is dependent on neither concentration nor reaction time. Accordingly, we propose a chemical equilibrium or a growth substrate limitation as the responsible mechanisms of the incomplete removal. Finally, Dokdonella could be the main acetaminophen degrader under activated sludge conditions, and non-antibiotic pharmaceuticals might still be toxic to relevant WWTP bacteria.     

Effect of activated carbon on the biological treatment of oil-water emulsions

Waste water, derived from the reprocessing of used emulsions or suspensions, contains high concentrations of emulsified mineral oil and stabilizers, as well as different additives that are needed during the treatment process. Two stirred-tank reactors and two fixed-bed reactors were used to study the biodegradation of these waste-water compounds during two-stage biological treatment. The waste water was first proceesed in an activated sludge reactor to remove easily biodegradable substances. The effluent from the first stage was treated in three parallel operating reactors: an activated sludge tank containing different amounts of powdered activated carbon (PAC, between 0 and 2%), an upflow anaerobic fixed-bed reactor and an aerobic fixed-bed reactor (trickling filter). The results from the continuous treatment were compared with laboratory batch experiments. About 60% of the influent TOC was reduced by the first activated sludge treatment. The removal efficiency increased to about 70% by using a second activated sludge stage. This degradation was comparable to the maximum degree of degradation measured in laboratory batch experiments. PAC addition to the second activated sludge tank resulted in increased degradation rates. The removal efficiency increased to about 76% when 0.1% PAC was added and to 96% with 1% PAC. The removal efficiency decreased to 84% when the proportion of PAC was further increased to 2%. Variations in the amount of PAC addition per unit influent volume in the range of 50 and 200 mg/l had no significant effect on the TOC removal. Degradation models based on the MONOD-type equation were found to be in close correlation with the results obtained from batch experiments. However, the biological removal rates measured in batch experiments did not reflect the removal capacity determined in continuous operating treatment systems.     

Removal of bisphenol A and diclofenac by a novel fungal membrane bioreactor operated under non-sterile conditions

Previous studies have confirmed significant removal of various trace organic contaminants (TrOCs) by white-rot fungal cultures under sterile batch test conditions. However, little is known about TrOC removal in continuous flow fungal reactors in a non-sterile environment. This study reports the removal of two TrOCs, namely, bisphenol A and diclofenac, by a fungal membrane bioreactor (MBR). Sterile batch tests with “active” (biosorption and biodegradation) and “chemically inactivated” (biosorption only) Trametes versicolor (ATCC 7731) confirmed biodegradation as the main mechanism for the removal of both compounds. An MBR inoculated with T. versicolor was operated in non-sterile conditions for a period of three months during which diclofenac and bisphenol A were continuously added to the synthetic wastewater. Relatively stable removal of bisphenol A (80–90%) and diclofenac (∼55%) was achieved by applying a hydraulic retention time of two days, at the bisphenol A and diclofenac loadings of 475 ± 25 and 345 ± 112 μg/L.d, respectively.     

Enhancement of Micropollutant Degradation at the Outlet of Small Wastewater Treatment Plants

The aim of this work was to evaluate low-cost and easy-to-operate engineering solutions that can be added as a polishing step to small wastewater treatment plants to reduce the micropollutant load to water bodies. The proposed design combines a sand filter/constructed wetland with additional and more advanced treatment technologies (UV degradation, enhanced adsorption to the solid phase, e.g., an engineered substrate) to increase the elimination of recalcitrant compounds. The removal of five micropollutants with different physico-chemical characteristics (three pharmaceuticals: diclofenac, carbamazepine, sulfamethoxazole, one pesticide: mecoprop, and one corrosion inhibitor: benzotriazole) was studied to evaluate the feasibility of the proposed system. Separate batch experiments were conducted to assess the removal efficiency of UV degradation and adsorption. The efficiency of each individual process was substance-specific. No process was effective on all the compounds tested, although elimination rates over 80% using light expanded clay aggregate (an engineered material) were observed. A laboratory-scale flow-through setup was used to evaluate interactions when removal processes were combined. Four of the studied compounds were partially eliminated, with poor removal of the fifth (benzotriazole). The energy requirements for a field-scale installation were estimated to be the same order of magnitude as those of ozonation and powdered activated carbon treatments.     

Oxidation of pharmaceutically active compounds by a ligninolytic fungal peroxidase

Pharmaceuticals are an important group of emerging pollutants with increasing interest due to their rising consumption and the evidence for ecotoxicological effects associated to trace amounts in aquatic environments. In this paper, we assessed the potential degradation of a series of pharmaceuticals: antibiotics (sulfamethoxazole), antidepressives (citalopram hydrobromide and fluoxetine hydrochloride), antiepileptics (carbamazepine), anti-inflammatory drugs (diclofenac and naproxen) and estrogen hormones (estrone, 17β-estradiol, 17α-ethinylestradiol) by means of a versatile peroxidase (VP) from the ligninolytic fungus Bjerkandera adusta. The effects of the reaction conditions: VP activity, organic acid concentration and H₂O₂ addition rate, on the kinetics of the VP based oxidation system were evaluated. Diclofenac and estrogens were completely degraded after only 5–25 min even with a very low VP activity (10 U l⁻¹). High degradation percentages (80%) were achieved for sulfamethoxazole and naproxen. Low or undetectable removal yields were observed for citalopram (up to 18%), fluoxetine (lower than 10%) and carbamazepine (not degraded).     

The effect of pharmaceuticals on the kinetics of methanogenesis and acetogenesis

In this study, the widely used anaerobic digestion model (ADM1) was used in order to simulate the inhibition of three pharmaceuticals, propranolol hydrochloride, ofloxacin and diclofenac sodium, on two groups of microorganisms, acetogens and acetoclastic methanogens, the most sensitive microorganisms groups involved in the anaerobic digestion process. The specific maximum consumption rate and saturation constant of acetate and propionate degraders were estimated through fitting the model to experimental data taken from continuous and batch experiments. A modified non-competitive inhibition function was used, and the inhibition constants were estimated using data from Batch experiments conducted at various concentrations of pharmaceuticals using enriched cultures with propionate and acetate degraders. It was found that propranolol hydrochloride was the most inhibitory pharmaceutical to both microorganisms groups.     

Baffled duckweed pond system for treatment of agricultural drainage water containing pharmaceuticals

The aim of the study is to assess the efficiency of a novel bioremediation system namely baffled duckweed pond (BDWP) system for the treatment of agricultural drainage water containing pharmaceuticals at different hydraulic retention times (HRTs). The removal efficiencies of acetaminophen (ACT), amoxicillin (AMX), and ampicillin (AMP) increased from 69.3 ± 8.6 to 87.3 ± 3.5%, from 52.9 ± 9.4 to 82.9 ± 5.2%, and from 55.3 ± 7.9 to 90.6 ± 2.8% at increasing the HRT from 6 to 8 days, respectively. However, ACT, AMX, and AMP removal efficiencies were slightly improved at increasing the HRT from 8 to 12 days. Diclofenac (DFC) removal efficiencies amounted to 56.6 ± 11.6, 55.7 ± 11.9, and 28.3 ± 12.9% at an HRTs of 12, 8, and 6 days, respectively. The results showed no relationship between the uptake/absorption of pharmaceuticals fractions and BOD₅/chemical oxygen demand (COD) ratio except ACT where R² was 0.84. The effect of COD/N ratio on the removal efficiency of pharmaceuticals fractions was slight. Additional removal of pharmaceuticals fractions and nitrification occurred in carrier sponge media situated in the last compartment of the BDWP.     

Impact of Anaerobic and Aerobic Processes on PolyChloroBiphenyl Removal in Contaminated Sewage Sludge

Aerobic and anaerobic biodegradation of six priority PCBs was investigated in continuous stirred tank reactors fed with naturally contaminated sewage sludge. Anaerobic and aerobic abiotic losses were higher for the lightly chlorinated PCBs but remained for all PCBs below 20%. Under strict methanogenic conditions, PCB removals were about 40% whatever PCB molecular weight or their degree of chlorination. However, considering abiotic losses, the heaviest PCBs were more efficiently anaerobically biodegraded probably because of higher dechlorination rates. The aerating sludge process enhanced removal of the lightest chlorinated PCBs from 40% up to 100%, while removal rates of the heaviest PCBs remained around 40%. Although the mesophilic aerobic process exhibits better removal efficiencies because of operating conditions, the results suggest that PCB biodegradation was strongly limited by their bioavailability in naturally contaminated sludge, under both redox conditions. Indeed, since PCB removal was closely linked to the solid reduction rates, PCB bioavailability was likely the limiting factor for biodegradation. As a consequence, the raw PCB concentrations (in mg kg^–1_{dry weight}) which are concerned by legislative procedures did not decrease sufficiently in both processes to reach a limit value fulfilling the current French/European regulation about PCB contents in sewage sludge before spreading on agricultural land.     

Anaerobic removal of linear alcohol ethoxylates

The present paper deals with a laboratory-scale study of anaerobic treatment of two commercial mixtures (LS2, LT7) of alcohol ethoxylates with 8-14 carbon atoms and 2 and 7 ethoxy groups. Tests were carried out in batch, with a 2 g l(-1) single dose, and in semibatch, with daily 0.2 g l(-1) doses. The behaviour of the tested mixtures was different: anaerobic sludge adsorption was the main removal process for LS2, while adsorption was less significant and biodegradation was more important for LT7. These differences appeared to be mainly related to the ethoxy portion length determining the extent of biodegradability and adsorption.     

Effect of Different Antibiotics and Non-Steroidal Anti-Inflammatory Drugs on the Growth of Lactobacillus casei Shirota

The purpose of this study was to investigate whether some non-steroidal anti-inflammatory drugs (NSAIDs) could cause inhibition of the growth of Lactobacillus casei Shirota (LcS) or whether this microorganism is able to use some of them as the sole carbon source, considering that the simultaneous consumption of NSAIDs and a dairy drink fermented with LcS could help to prevent the appearance or improve the healing of gastric ulcers. Acetylsalicylic acid (ASA), sodium acetylsalicylate (SAS), acetaminophen, sodium naproxen, and sodium ibuprofen were added as the sole carbon source to a basal medium and tested for biodegradation by LcS. The same NSAIDs were added in different concentrations to disks and plated on MRS Agar to test the possible inhibitory effect of these compounds on LcS. Also, the resistance of LcS to 12 different antibiotics was studied on MRS agar. None of the NSAIDs tested could be used by LcS as the sole carbon source at the assayed concentrations. In the case of the disk diffusion method, sodium naproxen showed inhibition zones for the 500-μg disks and sodium ibuprofen was inhibitory for the 250- and 500-μg disks. However, when the macrobroth dilution method was used, the growth of LcS was inhibited by ASA, SAS, acetaminophen, and sodium ibuprofen. This strain showed resistance to the antibiotics sulfamethoxazole with trimethoprim, pefloxacin, and gentamicin. This is the first study on the effect of NSAIDs on probiotic bacteria. The results of the biodegradation test indicate that the simultaneous consumption of NSAIDs and a dairy beverage with LcS is not likely to change the bioavailability of the drugs.     

Removal of pharmaceutical compounds in tropical constructed wetlands

The ability of tropical horizontal subsurface constructed wetlands (HSSF CWs) planted with Typha angustifolia to remove four widely used pharmaceutical compounds (carbamazepine, declofenac, ibuprofen and naproxen) at the relatively short hydraulic residence time of 2-4 days was documented. For both ibuprofen and naproxen, pharmaceutical compounds with low D_ow values, the planted beds showed significant (p < 0.05) enhancement of removal efficiencies (80% and 91%, respectively, at the 4 day HRT), compared to unplanted beds (60% and 52%, respectively). The presence of plants resulted in the removal of these pharmaceutical compounds from artificial wastewater. The more oxidizing environment in the rhizosphere might have played an important role, but other rhizosphere effects, beside rhizosphere aeration, appeared to be important also. Carbamazepine, considered one of the most recalcitrant pharmaceuticals, and declofenac showed low removal efficiencies in our CW, and this is attributable to their higher hydrophobicity. The fact that the removal of these compounds could be explained by the sorption onto the available organic surfaces, explains why there was no significant difference (p > 0.05) in their removal efficiencies between planted as compared to unplanted beds. No statistical significant differences (p > 0.05) were observed for the removal efficiencies of any of the pharmaceuticals tested for the 2-day HRT as compared to that corresponding to 4-day HRT. The rather efficient removal shown by the wetlands in this study (with HRTs of 2-4 days), indicates that such a CW system may be more practically used (with less land requirements) in tropical regions for removing conventional pollutants and certain pharmaceutical compounds from wastewater effluents.     

Biodegradation of Benzene in a Laboratory-Scale Biobarrier at Low Dissolved Oxygen Concentrations

A laboratory-scale permeable biobarrier exhibited high removal efficiencies of benzene at inlet concentrations of 0.4 to 35.1?mg/L and with a limited supply of dissolved oxygen. The supplied oxygen was less than the demand for a complete aerobic oxidation of benzene. Stainless steel pieces or granulated peat moss were used as packing material for microbial support in the biobarrier. Removal efficiencies ranged from 63.9% to 99.9% in the stainless steel-packed biobarrier and from 70.4% to 97.2% in the peat moss-packed biobarrier, while benzene elimination rate changed from 0.2 to 10.4?mg/L-d and from 0.1 to 3.7?mg/L-d in the two biobarriers, respectively. The consumption of sulfate and the presence of sulfate-reducing bacteria suggested the contribution of anaerobic metabolism in the biodegradation of benzene. The biodegradation of benzene under microaerophilic conditions (defined as dissolved oxygen concentrations <2?mg/L) was demonstrated during independent batch experiments. The maximum specific rate of benzene biodegradation with concentrations of 22.0 to 65.9?mg/L under microaero-philic conditions was 2.6 mg/mg biomass-d.     

The removal of Salmonella enteritidis in activated sludge

Salmonella destruction efficiencies of 99% were obtained after 10 h aeration at 15°C in a laboratory model of the activated sludge process. This study demonstrated that, in a batch process, the removal of salmonellas occurred in three phases. (i) By 4 h, 90% of the original inoculum had disappeared from the activated sludge, probably due mainly to predation by ciliated protozoa. The remaining 10% was distributed between the liquid phase (approximately 90%) and the sludge floc (approximately 10%). (ii) During the next 2 h this situation was inverted so that, by 6 h, more than 80% of the remaining salmonellas were then adsorbed to floc, leaving less than 20% in liquid suspension. (iii) From 6 h onwards there was a much slower decline of the remaining salmonellas attached to floc. The addition of dioctyl sodium sulphosuccinate after 4 h, inactivated the ciliated protozoa populations and completely eliminated the continued reduction of salmonellas from activated sludge observed previously.     

Biodegradation kinetic constants and sorption coefficients of micropollutants in membrane bioreactors

In order to elucidate the capability of biomass developed in membrane bioreactors (MBR) to degrade and sorb emerging micropollutants, biodegradation (k_biol) and sorption (k_sor) kinetic constants as well as solid–liquid partition coefficients (K_d) of 13 selected pharmaceutical and personal care products (PPCPs) were determined with MBR heterotrophic biomass adding a pulse (100 ppb of each compound) and following the liquid and solid phase concentrations over time. The results obtained were compared to literature data referring to conventional activated sludge (CAS) systems. Two experiments were performed: one in the MBR itself and the second one in a batch reactor with the same type and concentration of biomass as in the MBR. Overall, both biodegradation and sorption coefficients were in the same range as previously reported by other studies in CAS systems, indicating that MBR biomass does not show better capabilities for the biological degradation and/or sorption of PPCPs compared to the biomass developed in CAS reactors. Therefore, the higher PPCPs removal efficiencies found in MBRs are explained by the high biomass concentrations obtained at the long sludge retention times at which this type of reactors are usually operated.     

Effect of rhamnolipid on the aerobic removal of polyaromatic hydrocarbons (PAHs) and COD components from petrochemical wastewater

The removal efficiencies of 15 PAHs and some COD components (inert, readily degradable, slowly degradable and metabolic products) from a wastewater taken from a petrochemical industry treatment plant (İzmir, Turkey) have been determined using an aerobic completely stirred tank reactor (CSTR). Addition of rhamnolipid surfactant (15 mg l⁻¹) increased the removal efficiencies of PAHs and soluble COD from 72% and 90% to 80% and 99%, respectively. The rhamnolipid treatment caused a significant increase of 5- and 6-ring PAH degradation. The soluble COD removal efficiency was 93%, in CSTR reactors with rhamnolipid added. The inert COD removal efficiency was 60% in a CSTR reactor containing rhamnolipid. Batch tests showed that removal arising from the adsorption of the PAHs was low (between 1.88% and 4.84%) while the removal of PAHs from the petrochemical industry wastewater via volatilization varied between 0.69% and 5.92%. Low sorption capacity (K_p) values for refinery activated sludge (approximately 2.98 l g⁻¹) confirmed that bio-sorption was not an important mechanism controlling the fate of PAHs in aerobic CSTR reactors. Models proposed to simulate the PAH removal indicated that 94% of the PAHs were removed via biodegradation.     

Treatment of oilfield wastewater containing polymer by the batch activated sludge reactor combined with a zerovalent iron/EDTA/air system

Laboratory-scale experiments were conducted in order to evaluate the performance of a novel treatment process for oilfield wastewater based on combining chemical oxidation, performed by a zerovalent iron (ZVI), ethylenediamine tetraacetic acid (EDTA) and air process, with biological degradation, carried out in a batch activated sludge reactor. The influence of some operating variables was studied. The results showed that the optimum pretreatment conditions were 150 mg/L EDTA, 20 g/L ZVI, and a 180-min reaction time, respectively. Under these conditions, removal efficiencies for hydrolyzed polyacrylamide (HPAM), total petroleum hydrocarbons (TPH), and chemical oxygen demand (COD) were 66%, 59%, and 45%, respectively. During the subsequent 40 h of bioremediation, the concentrations of HPAM, TPH, and COD were decreased to 10, 2 and 85 mg/L, respectively. At the end of experiments, the total removal efficiencies of HPAM, TPH, and COD were 96%, 97% and 92%, respectively.     

Application of redox mediators to accelerate the transformation of reactive azo dyes in anaerobic bioreactors.

Azo dyes are nonspecifically reduced under anaerobic conditions but the slow rates at which reactive azo dyes are converted presents a serious problem for the application of anaerobic technology as a first stage in the complete biodegradation of these compounds. As quinones have been found to catalyze reductive transfers by acting as redox mediators, the application of anthraquinone-2,6-disulfonic acid (AQDS) during continuous anaerobic treatment of the reactive azo dye, Reactive Red 2 (RR2), was evaluated. A mixture of volatile fatty acids was used as the electron-donating primary substrate. Batch experiments demonstrated that AQDS could increase the first-order rate constant of RR2 reductive cleavage by one order of magnitude. In the continuous experiment, treatment of RR2 containing synthetic wastewater in a lab-scale upflow anaerobic sludge blanket (UASB) reactor yielded low dye removal efficiencies (<30%). Consequently, severe toxicity problems occurred, eventually resulting in almost complete inhibition of the methanogenic activity. Addition of catalytic concentrations of AQDS (19 microM) to the reactor influent caused an immediate increase in the dye removal efficiency and recovery of biological activity. Ultimately, RR2 removal efficiency stabilized at 88%, and higher AQDS loads resulted in higher RR2 removal efficiencies (up to 98% at 155 microM AQDS). Examination of the RR2 decolorizing properties of dye-adapted reactor sludge and of nonadapted reactor seed sludge revealed that RR2 decolorization was principally a biologically driven transfer of reducing equivalents from endogenous and added substrates to the dye. Hydrogen, added in bulk, was clearly the preferred electron donor. Bacteria that couple dye decolorization to hydrogen oxidation were naturally present in seed sludge. However, enrichment was required for the utilization of electrons from volatile fatty acids for dye reduction. The stimulatory effect of AQDS on RR2 decolorization by AQDS-unadapted sludge was mainly due to assisting the electron transfer from endogenous substrates in the sludge to the dye. The stimulatory effect of AQDS on RR2 decolorization by sludge from the AQDS-exposed reactor was, in addition, strongly associated with the transfer of electrons from hydrogen and acetate to the dye, probably due to enrichment of specialized AQDS-reducing bacteria.     

Lysinibacillus sphaericus proved to have potential for the remediation of petroleum hydrocarbons

In this study, the ability of Lysinibacillus sphaericus to degrade aromatic hydrocarbons as well as complex hydrocarbon mixtures, such as diesel oil and oily sludge, was evaluated. L. sphaericus was able to grow when toluene, naphthalene, or phenanthrene were used as a sole carbon source in minimal salt medium. Removal efficiencies of up to 95% were found for C₁₀-C₂₈ hydrocarbons in the biodegradation assays of diesel oil. The biodegradation of oily sludge was evaluated in landfarming-like experiments in the open air and in completely covered containers in the field. After 50 days of treatment, the removal efficiency of total petroleum hydrocarbons in open-air and closed assays was of 84.1% and 60.1%, respectively. Furthermore, L. sphaericus was able to degrade volatile hydrocarbons (benzene, toluene, ethylbenzene, and phenol) in the headspace of closed containers, preventing the emission of these compounds to the atmosphere. L. sphaericus was herein proposed as a promising candidate to be used in bioremediation strategies of petroleum hydrocarbons.