Enhanced removal of emerging micropollutants by applying microaeration to an anaerobic reactor

The present paper aimed to evaluate the impact of microaeration on both the removal performance of some emerging micropollutants (pharmaceuticals, hormones, and bisphenol A) and the microbial community structure of an anaerobic reactor treating synthetic wastewater. Under anaerobic conditions, the removal efficiencies of the micropollutants were very low (< 10%). However, the microaeration (1.0 mL air·min -1 at 27 °C and 1 atm, equivalent to a Q AIR /Q INF ratio of 0.1) expressively improved the removal efficiencies of all compounds (> 50%). Therefore, supplementing anaerobic reactors with low amounts of oxygen seems to be an interesting strategy to enhance the removal of the micropollutants tested. However, further studies should be carried out with other compounds in order to evaluate the wide applicability of microaeration to different classes of micropollutants in lab- and full-scale treatment systems. Concerning the microbiota structure, both bacterial and archaeal communities were not compromised by the different operational conditions and preserved their functional organization with high richness during the whole experiment.


INTRODUCTION
Several emerging micropollutants from different classes (e.g. pharmaceuticals and hormones) are consumed every year worldwide. Such pharmaceutical compounds include antipyretics, analgesics, lipid regulators, antibiotics, antidepressants, chemotherapeutics, contraceptives, among others (YANG et al., 2017). In addition, some compounds, such as bisphenol A (BPA), which is mainly used in the production of polycarbonate plastics and epoxy resins, also have estrogenic activity (ZIELIŃSKA et al., 2014). Therefore, the occurrence of these micropollutants in aquatic environments has brought impacts on fauna, flora, and human health to light (TAMBOSI et al., 2010).
Adverse effects caused by these emerging micropollutants include aquatic toxicity, increase in pathogenic bacteria resistance, genotoxicity, increase in breast and prostate cancer incidence, endometriosis, and other endocrine disorders (AQUINO; BRANDT; CHERNICHARO, 2013;KÜMMERER, 2010). Thus, the development of processes that can promote the effective removal of micropollutants, along with DOI: 10.1590 other priority pollutants, is an emerging issue in science and environmental engineering. These processes need to reach certain goals, such as higher efficiency, compliance with environmental requirements, more compact units that operate with greater flexibility and efficiency, and lower installation and operational costs (AQUINO; BRANDT; CHERNICHARO, 2013).
There are biological and non-biological processes (physical, chemical, and physicochemical) for removing these compounds from environmental water matrices. The non-biological methods include advanced oxidation processes, ozonation, nanofiltration, reverse osmosis, and adsorption on zeolite or activated carbon (DE LA CRUZ et al., 2012;VIDAL et al., 2015). However, these techniques incur high installation and operational costs. Furthermore, non-destructive techniques (e.g. physical) require auxiliary processes intended to adsorb, degrade, or dispose of the pollutants previously extracted (AQUINO; BRANDT; CHERNICHARO, 2013;PESSOA et al., 2014).

Some investigations into micropollutants removal by anaerobic
reactors have been carried out, but their removal efficiencies are much lower than those of aerobic treatment systems (ALVARINO et al., 2016;DE GRAAFF et al., 2011;JOSS et al., 2004).
Recent studies have shown that adding low oxygen concentrations (microaeration) to anaerobic systems could improve the initial degradation of recalcitrant compounds, such as monoaromatic hydrocarbons (BTEX) SIQUEIRA et al., 2018). Nevertheless, to the best of the authors' knowledge, there has been no investigation into microaeration of anaerobic reactors for micropollutants removal.
Hence, the present paper aimed to assess the impact of microaeration on both the removal performance of some emerging micropollutants (the natural estrogens estrone (E1) and estradiol (E2), the synthetic estrogen ethinylestradiol (EE2), the anti-inflammatory diclofenac (DCF), the antibiotics sulfamethoxazole (SMX) and trimethoprim (TMP), and the xenoestrogen bisphenol A (BPA)) and the microbial community structure of an anaerobic reactor treating synthetic wastewater.

Experimental set-up
The continuous flow experiment was carried out in an upflow anaerobic sludge blanket (UASB) reactor, with a working volume of 3.7 L, made from PVC tubes and connections for sewage. The reactor was inoculated with anaerobic sludge (~60 g SSV·L -1 ) from a mesophilic internal circulation (IC) reactor of a brewery (Horizonte, Ceará, Brazil).
The influent was kept under refrigeration throughout the experiment (~5 °C) to avoid degradation, and the reactor was fed by a peristaltic pump (Minipuls 3, Gilson, USA) through a Tygon ® flexible tubing (Cole-Parmer, USA), at an average flow rate of 14 L·d -1 (TDH ≈ 7 h).
A dosing pump (Concept ProMinent Dosiertechnik GmbH, Germany) was used to recirculate the effluent (at 0.7 L·h -1 ) in order to improve mass transfer, avoid preferential paths, and facilitate the release of biogas bubbles, thus preventing biomass loss due to the piston effect.
The microaeration was introduced into the reactor at its feeding line from a gas cylinder containing synthetic air (20% O 2 :80% N 2 ), by using a mass flow controller (Cole Parmer, USA), at an airflow rate of 1.0 mL·min -1 (at 27 °C and 1 atm). This airflow rate corresponds to a microaeration rate (MR) of approximately 0.10, which is calculated as the ratio between the airflow rate and the influent flow rate of the reactor (Q AIR /Q INF ).
The produced biogas was collected and quantified by the liquid displacement method and characterized by gas chromatography as specified in the chemical and chromatographic analyses section.

Synthetic wastewater composition
The synthetic wastewater was prepared weekly by dissolving in potable tap water a mixture of the following micropollutants

Experimental procedure
The experiment was run in three periods (Table 1). In period I (acclimatization), the reactor was operated under anaerobic conditions and fed with micropollutant-free wastewater. Therefore, ethanol (1.0 g COD·L -1 ) was the only carbon and energy source. In period II, the micropollutants were added to the synthetic wastewater in order to assess the removal performance of the reactor under anaerobic conditions. The concentration of the micropollutants (~230 µg·L -1 of each compound) used in this study was in accordance with that observed in domestic wastewaters (PESSOA et al., 2014). Finally, in period III, a microaeration flow rate of 1.0 mL·min -1 of synthetic air (at 27 °C and 1 atm) was introduced into the reactor at its feeding line (Q AIR /Q INF = 0.1) to evaluate the micropollutants removal performance under microaerobic conditions. This airflow rate was set based on previous studies on microaerobic BTEX removal SIQUEIRA et al., 2018).
The transition between experimental periods occurred after checking the stability of effluent chemical oxygen demand (COD) and micropollutants concentrations in the last five data (variation up to 10%).

Chemical and chromatographic analyses
COD and pH were determined according to APHA (2005), whereas the pharmaceuticals and hormones were determined by high-performance liquid chromatography with diode array detection (HPLC-DAD) according to Vidal et al. (2015). The biogas was characterized in terms of air (O 2 + N 2 ), CH 4 , and CO 2 by gas chromatography with thermal conductivity detection (GC-TCD) (GC-17A, Shimadzu Corporation, Japan) according to Firmino et al. (2015).

Microbial community analysis
To evaluate the microbial community structure (functional organization, richness, and diversity), sludge samples, including the inoculum, were withdrawn from the reactor at the end of all experimental periods and frozen at -20 °C, until the genomic DNA was extracted using a fast extraction kit (Biomedicals, USA) following the manufacturer protocol. The DNA concentration (0.2 to 2 mg·L -1 ) was determined by spectrophotometry with a Nanodrop 2000 (Thermo Fisher Scientific, USA).
Bacterial and archaeal community structure was analyzed by polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) according to Sousa et al. (2016), as follows. The 16S rRNA gene

Statistical methods
In order to compare the reactor performance in the three periods, nonparametric tests were used (Mann-Whitney and Kruskal-Wallis). The results were considered statistically different when p ≤ 0.05.

Removal of emerging micropollutants
After the acclimatization (period I), the reactor started to be fed with the micropollutant-containing wastewater (period II). As expected, under anaerobic conditions, the average removal efficiencies of all compounds were very low (< 10%) (Table 2). Therefore, adsorption is not an important mechanism for removal of micropollutants under anaerobic conditions (HARB et al., 2019). Among the estrogens, E1 presented the highest average removal efficiency (9%), whereas E2 and EE2 showed average values near 5% (Table 2).
According to Joss et al. (2004) andDe Mes et al. (2007), E2 presents lower removal efficiencies than E1 in anaerobic treatment systems because low redox potential values favor the reduction of E1 to E2.
EE2 is more recalcitrant due to steric hindrance, i.e., the ethinyl group at position 17 does not allow the formation of a ketone (as observed in E2), thus its removal efficiency is lower than that of E2 (CZAJKA; LONDRY, 2006). According to Aquino, Brandt, and Chernicharo (2013), the low anaerobic biodegradability of emerging micropollutants is probably due to the presence of phenolic aromatic rings in their structures, which are more difficult to degrade in the absence of dissolved oxygen.
In period III, microaeration was applied to the reactor at its feeding line, and the average removal efficiencies of all compounds increased from below 10% to above 50%. This improvement is significant when compared to the anaerobic period (Table 2). Therefore, supplementing anaerobic reactors with low amounts of oxygen seems to be an interesting strategy to enhance the removal of the micropollutants tested.
However, further studies should be carried out with other compounds in order to evaluate the wide applicability of microaeration to different classes of micropollutants in lab-and full-scale treatment systems.
De Mes et al. (2007) identified an increase of up to 40% in the estrogens removal efficiency (E1, E2, and EE2) when a downflow hanging sponge (DHS) reactor was operated as a microaerobic post-treatment for blackwater anaerobically treated by a UASB reactor. According to the authors, the microaeration of the post-treatment intensified the degradation of these compounds. Joss et al. (2004) demonstrated that the biological removal of some estrogens depended on the biomass activity and the redox potential in the treatment systems, i.e., the presence of oxygen is an important factor for the removal of these compounds. Finally, it is noteworthy that COD removal efficiencies were higher than 90% during the whole experiment (Table 3). Despite the slight reduction in the average values of COD removal efficiency and methane production in period III when compared to the previous periods, there is no statistically significant difference among all experimental periods (Table 3). Therefore, microaeration did not alter the organic matter removal capacity of the reactor. These results corroborate those by Siqueira et al. (2018), who did not found any significant difference in COD removal (~80.5%) after microaerating, at different airflow rates (0.5-2 mL·min -1 ), an anaerobic reactor fed with BTEXcontaminated water.

Microbial community structure
The effect of the different operational conditions on the bacterial and archaeal communities of the reactor can be observed in their corresponding DGGE profiles (Figure 1), from which the ecological parameters Rr and Fo were calculated (Table 4) to evaluate the changes in the microbiota.
After period I, when ethanol was the only carbon source, the richness of the bacterial community increased expressively (from 8, low Rr, to 91, high Rr), whereas its evenness remained high (Fo < 30%) (Table 4). These parameters indicate the development of a community formed by groups of generalist organisms without specific dominance, which might have been due to the carbon source used (ethanol), a simple and easily degradable substrate.

The introduction of the micropollutants (period II) reduced both
Rr and Fo of the bacterial community (Table 4). Nevertheless, its richness and evenness remained high according to Marzorati et al. (2008).
Therefore, the environment maintained a broad carrying capacity, being considered very habitable, as the microbial diversity remained high (MARZORATI et al., 2008).
In period III, Rr increased considerably, whereas Fo presented a slight decrease (Table 4). Therefore, the microaeration might have positively affected the bacterial community, increasing its diversity, i.e., some monooxygenase-producing populations might have grown, favoring the micropollutants biotransformation as observed in previous investigations into microaerobic BTEX removal SIQUEIRA et al., 2018). However, no specific group showed dominance, as the community evenness remained high (Fo < 30%) ( Table 4).
As for the archaeal community, no remarkable changes were found among the inoculum and all experimental periods, in which richness and evenness remained very high (MARZORATI et al., 2008).
Consequently, the operational changes did not cause sufficient stress to modify the microbiota structure, and its functionality was preserved during the whole experiment. Probably, the structural configuration of granular sludge, in which the facultative or microaerophilic bacteria (in the outer layers) protect the strictly anaerobic archaea (in the granule core) from oxygen, and the low retention time of oxygen in the sludge blanket might have contributed to maintain the archaeal community .
In general, the introduction of the micropollutants and microaeration (periods II and III, respectively) did not compromise the structure of the bacterial and archaeal communities, as, according to Marzorati et al. (2008), evenness and richness remained high during the 200 days of operation (Table 4).

CONCLUSIONS
Under anaerobic conditions, the removal efficiencies of the emerging micropollutants were very low (< 10%). However, the microaeration expressively improved the removal efficiencies of all compounds (> 50%). Therefore, supplementing anaerobic reactors with low amounts of oxygen seems to be an interesting strategy to enhance the removal of the micropollutants tested.