The study was conducted to investigate the effect of sludge retention on bacterial community composition of membrane bioreactor (MBR) treating synthetic hospital wastewater. The removal of four pharmaceuticals, namely carbamazepine, estradiol, venlafaxine, and ibuprofen in MBR, was studied at varying sludge retention time (SRT) duration of 100, 45, and 15 days and hydraulic retention time (HRT) of 18 h. The removal of ibuprofen and estradiol was constant at varying SRT; however, a negligible removal of carbamazepine and low removal of venlafaxine was observed (< 20%). The study suggested that the SRT of 45 days in MBR could provide maximum treatment efficiency via decreasing membrane clogging. The effect of sludge age and pharmaceutical presence on the bacterial community was investigated via high-throughput sequencing. The study reveals that the variation in SRT affects the dynamics of the bacterial community significantly. For instance, the dominant bacterium Caldimonas of SRT 100 was disappeared at lower SRTs. Moreover, the profile of the dominant genus of the SRTs varied greatly from each other.
The presence of emerging contaminants, such as pharmaceuticals, microfibers, personal care products in the aquatic environment, was extensively reported by many studies [1, 2]. The occurrence and constant release of these emerging pollutants in the environment from different sources, including industries, hospitals, and health care homes, animal farms, and households, are a matter of concern due to their potential eco-toxicological effects on living organisms. Pharmaceuticals are developed and designed for curing and preventing disease; however, their partial metabolism in human and animal bodies leads to their introduction in the environment, which makes pharmaceutical compounds emerging contaminants. The presence of a different class of pharmaceuticals, which includes antibiotics, antidepressants, analgesic, antiepileptic, diuretic medicine in a different compartment of the environment, was documented . Andreu et al.  reported the presence of seventeen pharmaceuticals in the coastal wetland of Oliva and Pego municipalities of Spain. Among the thirty-four sampling sites of the wetland, the presence of pharmaceuticals was detected in the thirty-three sites with the concentration ranging from 1.2 to 63 ng L−1 . In Canada in south central region of Quebec, a study reported the presence of ibuprofen and carbamazepine with a concentration ranging from 30 to 97 ng L−1 in drinking water  whereas the concentration of these two pharmaceuticals (ibuprofen and carbamazepine) in industrial wastewater ranges between 50,000 to 230,000 ng L−1 . A study on the presence of pharmaceuticals in municipal wastewater treatment plants (WWTP) located in Quebec, Canada reported the elevated concentration of venlafaxine and carbamazepine i.e., 1096 and 217 ng L−1, respectively, in municipal wastewater . Till now, all of the pharmaceuticals, which are developed or produced so far (including veterinary drugs) are detected either in wastewater or in natural water bodies .
The implication of pharmaceuticals contaminants in the environment involves, development of antibiotic or multidrug-resistance pathogen (bacteria, fungi), interference in the reproductive system, and disruption of organs of non-targeted organisms . A study reported the decline in vulture population in India is due to the presence of diclofenac in the food . However, In-spite of numerous investigations, the data on the specific eco-toxic effect of each pharmaceutical on living organisms in the different environmental sites are limited. Moreover, the synergistic effect of different pharmaceuticals, metabolites, metals and other chemical contaminants present in the environment raises a concern for the ecosystem and human health. Yan et al., (2020)  summarized the US EPA (Environment Protection Agency) guidelines for hospital effluent discharge. As per the EPA guidelines, the biological oxygen demand (BOD) and total suspended solids (TSS) values in hospital wastewater should not exceed 41 and 55.6 kg/1000 beds/day . Based on the available literature, there are no guidelines available for the discharge limit of pharmaceutical compounds present in hospital wastewater.
From the last few decades, attention has been paid to the removal of pharmaceuticals from wastewater using different wastewater treatment strategies. Various biological and physicochemical treatment options, such as the activated sludge method, aerobic/anaerobic membrane bioreactor process, advanced electro-oxidation process, photo-Fenton process, and many more, have been investigated for the treatment of wastewater containing pharmaceuticals. However, the different treatment option has its pros and cons. Radjenvoic et al.  showed that despite the good quantification limit of pharmaceuticals detection (~ 1.1–84.7 ng L−1), the removal of several pharmaceuticals (such as diclofenac, mefenamic acid, gemfibrozil) in the conventional activated sludge process was insignificant, i.e. ≥ 25%. Moreover, the generation of excessive amounts of sludge (containing pharmaceuticals) adds to the problem of sludge disposal in the environment.
Among the different treatment options, the membrane bioreactor (MBR) technique has been extensively studied for the efficient removal (≥ 95%) of pharmaceuticals [3, 11]. Tiwari et al. , suggested that the biological treatment of wastewater using membrane bioreactor technique could provide efficient removal (i.e., > 95%) of pharmaceuticals. The membrane filtration process removes the settling and clarifying step of the conventional process, and low sludge production due to retention of biomass within the reactor provides an additional benefit over the conventional activated sludge process. The sludge retention in MBR prevents the dissemination of antibiotic-resistance bacteria (ARB) and antibiotic-resistance genes (ARGs) and provides a longer degradation time to microbiome MBR for the removal of pharmaceuticals. The principal removal mechanism of pharmaceuticals in MBR is bio-degradation , which implies that the microbial community of MBR has a key role in the removal of pharmaceuticals from wastewater.
The operating parameters, such as hydraulic retention time (HRT), sludge retention time (SRT), pH, temperature, and reactor configuration, determine the performance of the MBR system. Among those, HRT and SRT are the two important parameters that determine the treatment efficiency of the MBR by controlling microbial community structure and membrane fouling rate. The previous study reveals that the change in HRT determines the performance of the MBR by reducing the clogging at a longer hydraulic retention time (HRT) . The SRT, which determines the time during which sludge is retained within the reactor, influences not only the excess sludge discharge rate but also affects the sludge characteristics. For instance, shorter SRT results in the washout of slow-growing microbes, and the micro-organisms having a fast growth rate could proliferate in the reactor. On the other hand, long SRT facilitates the proliferation of micro-organisms, having a slow growth rate and endogenous respiration, which reduces the sludge discharge. Moreover, the removal of pharmaceuticals could have enhanced at longer SRT, as high biomass concentration at longer SRT reduces the food to micro-organism ratio, which might lead to the degradation of complex organic compounds (pharmaceuticals) by micro-organisms to fulfill their energy demands.
In this regard, we conducted a study to optimize the SRT of the submerged membrane bioreactor (MBR) system for the treatment of synthetic wastewater spiked with pharmaceuticals. Pharmaceuticals are biologically active compounds and known to have a profound effect (bacteriostatic, bactericidal, community shift) on the microbial community. The previous investigation reported that the MBR at an optimized HRT of 18 h was able to remove pharmaceuticals; however, the presence of pharmaceuticals changes the bacterial community of MBR . Thus, the presence of pharmaceuticals and change in SRT could influence the dynamics of the bacterial community of biological treatment. Moreover, the information on the spatial distribution of bacterial communities in the environment, specifically in the presence of pharmaceuticals, is limited [1, 3, 12]. Thus, the present work was in continuation with the previous work of the same research group to optimize the SRT of the MBR for the efficient removal of pharmaceuticals. The study also investigates the bacterial community of the MBR microbiome to understand the shift and role of micro-organism in the removal of pharmaceuticals.
Materials and methods
Preparation of synthetic wastewater
The synthetic wastewater equivalent to hospital wastewater was prepared using the composition adapted from Tiwari et al., . The synthetic wastewater contained (NH4)2SO4 -353.57; KH2PO4—92.19; MgSO4 -34.68; CaCl2.2H2O—22.10; FeCl3—11.63; CuSO4—0.07; Na2MoO4.2H2O—0.10; MnSO4.H2O—0.12; ZnCl2—0.23; CoSO4.7H2O -0.48; Na2CO3- 428.60; C6H12O6- 938.35, C6H12O6—1000 mg L−1. The study targeted the removal of four pharmaceuticals namely, ibuprofen (IBF), carbamazepine (CBZ), estradiol (EE2), and venlafaxine (VNF) at SRT of 15, 45, and 100 days. Literature investigation was performed to choose the concentration of pharmaceuticals based on their presence in real hospital wastewater [3, 13]. The concentration of pharmaceutical pollutants utilized in the study was: IBU—10 µg L−1, CBZ—10 µg L−1, EE2—10 µg L−1, and VNF—0.2 µg L−1. The detailed preparation and storage condition of stock solution of pharmaceuticals and synthetic wastewater were described in Tiwari et al. . All the chemicals (purity grade of ~ 99%) were purchased from Sigma–Aldrich Canada Ltd. (Oakville, ON, Canada).
The submerged membrane bioreactor (MBR) containing a hollow fiber membrane (ZW-1, Zenon Environmental Inc., Canada) pore size of 0.04 µm and filtering surface area of 0.047 m2 was operated in continuous mode for treatment of wastewater. The hollow fiber membrane was placed vertically inside the reactor and connected with the peristaltic pump for the filtration. The filtration cycle of the 50 s followed 10 s of backwashing was performed to prevent rapid clogging of the membrane. Air was supplied continuously within the membrane module and from the bottom of the reactor to maintain the aerobic condition. The MBR was inoculated with the activated sludge of municipal wastewater treatment (Victoriaville, Quebec). Initially, synthetic wastewater was fed as an influent in MBR for the development of biomass, followed by the addition of pharmaceuticals after 60 days of operation. The detailed study on MBR setup, biomass development, and pharmaceutical addition was described in the previous investigation Tiwari et al., . The operating conditions of MBR, i.e., pH, temperature, and HRT, were 7 ± 0.5, 20 °C ± 1 °C and 18 h, with a permeate flow rate of 10.21 L/m2 h, respectively.
Three SRTs (15, 45, and 100 days) were chosen to study the impact of SRT on pharmaceutical removal and bacterial community shift within the MBR. The SRT was maintained within the reactor by withdrawing a calculated amount of sludge daily.
Bacterial community analysis
The bacterial community analysis was performed by isolating the total genomic DNA from sludge sampled during the varying SRT condition. Prior to DNA isolation, sludge was centrifuged and washed twice to remove humic acid and other impurities. The gDNA was isolated using a Power soil DNA isolation kit [MoBio]. For each collected sludge sample, the DNA was extracted thrice separately and pooled together, and then subjected to 16 s rRNA sequencing in duplicates.
The 16 s rRNA gene amplification was performed using a universal primer (341F and 805R). The sequence library preparation and Illumina Miseq were performed. The raw sequence read was analyzed using Usearch 10 tool with a few exceptions. Primarily, the paired end was merged followed by primer removal and de-replication. Singleton was removed, and UPARSE algorithm was used for OTU clustering. The taxonomic affiliation of OTU was predicated by the k-mer similarity of OTU representative sequences to the RDP (Ribosomal Database Project) v.16 training set of 16S rRNA gene. The detailed procedure of analysis of raw sequence data was provided in Tiwari et al. .
The wastewater and treated wastewater samples from MBR were collected regularly for assessment of MBR performance (in terms of removal of pharmaceutical, COD, N-NH4, and P-PO4). The pharmaceutical analysis was performed using liquid chromatography and tandem mass spectrometry analysis (LC–MS/MS) (Thermo Scientific TSQ Quantiva Triple-Stage Quadrupole Mass Spectrometer, Germany). The detailed procedure of LCMS-MS analysis was described in Tiwari et al., . The SS, VSS, and COD analysis was performed using protocol CEAEQ, 2015–2016 [14, 15]. The online colorimetric assayed (QuikChem® 10-107-06-2 Method-B and Method QuikChem® Method 10-115-01-1-Q LACHAT Instrument, USA) was carried out for N-NH4 and P-PO4 estimation. The limit of detection and limit of quantification of all the procedures are provided (SL Table -1).
Results and discussion
Treatment efficiency of MBR
The study was conducted to evaluate the effect of SRT on the removal of pharmaceutical pollutants. The optimization of SRT in MBR was required not only to maximize the removal of pharmaceuticals pollutants but also to decrease the membrane clogging rate. The operation of MBR at a certain SRT could increase or decrease the production of extracellular polymeric substances (EPS), which is the major contributor to membrane clogging. The microbial community responsible for EPS secretion tends to adhere to membrane surface, thus resulting in cake formation . An increase in soluble EPS concentration at a shorter SRT of 20 days in MBR treating dairy effluent has been reported. However, at a longer SRT of 80 days, an increase in colloidal EPS concentration due to the cell lysis was observed . The study also suggested that the longer sludge age could have enhanced degradation of polysaccharides and protein by slow-growing microbes leading to less production of biopolymer. Moreover, a study reported that the presence of EPS influences sulfamethoxazole adsorption . However, an upper limit of SRT should be defined depending on the wastewater characteristics to reduces the colloidal EPS production and subsequently to prevent the membrane fouling. Therefore, the three SRT durations of 100, 45, and 15 days were chosen to study MBR operation for a period of approximately 150, 130, and 90 days, respectively.
Initially, an SRT of 100 days was examined for the removal of pharmaceuticals in MBR at an HRT of 18 h. The longer SRT of 100 days resulted in maximum SS and VSS concentration of 15.8 g L−1 and 13. 3 g L−1, respectively. The decrease in SRT from 100 to 45 days and subsequently from 45 to 15 days decreased the SS and VSS concentration gradually. The average concentration of SS and VSS in MBR at SRT of 45 and 15 days was 8.7 (SS) and 6.66 (VSS) g L−1, and 6.76 (SS) and 4.8 (VSS) g L−1, respectively (Fig. 1). Furthermore, a slight decrease in SS and VSS concentration at SRT of 100 days was observed with the operation time (Fig. 1). This decrease in SS and VSS concertation might be due to endogenous respiration and loss of sludge during the membrane cleaning. In-spite of endogenous respiration, the longer SRT of 100 days resulted in a high concentration of SS and VSS in MBR. The COD, N-NH4, and P-PO4 removal in MBR decreased with a decrease in SRT (Fig. 1). Despite the reduction in MBR treatment efficiency at 15 days SRT, the MBR was able to remove > 75% of COD and > 70% of N-NH4. However, the P-PO4 removal was < 47% at SRT of 100 days and decreased to 16% at 15 days SRT (Fig. 1). The decrease in removal efficiency of COD, N-NH4, and P-PO4 could be attributed to the low biomass concentration at shorter SRT. These findings are in agreement with the other studies, such as Fadel et al. , reported a decrease in removal efficiency of COD, BOD, total phosphorus, and PO43− in MBR with the reduction in SRT.
The removal of IBF and EE2 in MBR was consistent at varying SRT and accounted for > 90% and > 95%, respectively. The consistency in the removal revealed that, even though the biomass concentration reduced with a decrease in SRT, it provided adequate SS concentration for IBF and EE2 removal. The removal of CBZ was negligible in MBR at SRT of 100 d, a negative removal of CBZ (~ -25%) was observed. Interestingly, the negative removal of CBZ decreases with SRT and accounts for < − 10% at SRT of 45 days and completely stopped at SRT of 15 days and a positive removal of ~ 10% was observed (Fig. 2). The decrease in negative removal confirms the assumption that during the treatment, a small fraction of CBZ, which was initially sorbed on the sludge gets desorbed with time and contributes to negative removal at a longer SRT duration . Since the reduction in SRT increases the amount of sludge withdrawn from MBR, the CBZ residues sorbed on the sludge were removed with the discharge of excess sludge. The removal of VNF fluctuates considerably during the entire study period and ranges between 40 and 10% at varying SRT. It was difficult to predict the co-relation between the SRT and VNF removal in MBR as the average removal of VNF accounts for 19.5% at all three SRTs. The SRT did not have much effect on the removal of IBF, EE2, and VNF in MBR. This could be due to high biomass concentration and low pharmaceutical concentration (in µg L−1 range), which provide adequate treatment conditions. Bo et al.  reported that in the wastewater treatment plant, the elimination of ibuprofen was governed by the biodegradation process, the study further reported that the sludge microbiome was able to degrade 60 µg L−1 of ibuprofen spiked in treated municipal wastewater completely in 2 h in the batch experiment. Fernandez-Fontaina et al.,  studied the removal mechanisms of ibuprofen in activated sludge processes operated at varying SRT, ranging from 10 to 170 days. The author further states that the bio-degradation is the major removal mechanism and varying SRT did not affect the removal efficiency of ibuprofen and aerobic condition is sufficient for complete removal of ibuprofen even at a short SRT duration of 10 days. A study suggested that the removal of CBZ was independent of SRT and even an SRT of 275 days did not enhance the removal of carbamazepine above 10% in wastewater treatment plant . The researchers concluded that the persistence of carbamazepine might be due to its complex heterocyclic structure, hydrophilicity, and low adsorption coefficient [1, 23]. Rúa-Gómez et al.  reported an increase in the removal of venlafaxine from 37 to 85% after the addition of powder activated carbon (PAC) in MBR. This finding indicated that the PAC addition increased the sorption of VNF. However, the study on the removal mechanism of VNF in the wastewater treatment plant is limited. Thus, it was difficult to predict the fate of VNF in the biological treatment of wastewater. The high sludge adsorption coefficient of estradiol (~ 2.3–2.83) validates its high removal in MBR .
Bacterial community analysis
The bacterial community analysis was categorized into three groups depending upon the varying SRT duration. During the entire period of the study, the pharmaceuticals were continuously fed into the MBR at a concentration of 10 µg L−1 for IBF, CBZ, and EE2 and 0.2 µg L−1 for VNF at varying SRT of 100, 45, and 15 days, which named as Phase 1, Phase 2, and Phase 3, respectively. The operating time of each phase was provided (SL Table 2). Fifty-five samples corresponding to different SRT conditions were used to study the bacterial community structure of MBR biomass. The 16 s rRNA gene amplicon was used to perform Illumina Mi-Seq sequencing for bacterial community analysis. Figure 3 presents the distribution of phylum at varying SRT. The phylum distribution reveals that although the proteobacteria were the dominant phylum in all the phases, their relative abundance increases with a decrease in SRT, i.e., 63.9 at SRT of 100 days to 74.44% at 15 days SRT. The dominance of proteobacteria in MBR was consistent with the other studies on soil microbial community analysis [1, 25]. Spain et al.  validated that the proteobacteria were the most abundant phylum, which has an important role in the carbon, nitrogen, and sulfur cycle and consist of varied physiological, morphological, and metabolic diversity. Following proteobacteria, the second-most abundant phyla in phase 1 and phase 3 was Bacteroidetes, and actinobacteria in phase 2. The abundance of Bacteroidetes in phase 1 (~ 21.91%) and phase 3 (~ 10.71%) can be explained by the fact that they can inhibit the growth of competitor bacterial strains by secreting antibacterial protein (bacteriocin) . The capacity of Bacteroidetes to consume polymers, tendency to attach with particles for growth, and gliding motility which allows movement of bacteria along the surface, facilitates its proliferation and thereby its abundance in the environment . Actinobacteria are known for the production of secondary metabolites, such as enzymes, antibiotics, antitumor, and anti-inflammatory compounds. The presence of antibiotic-producing genes in actinobacteria implies that it has the machinery to confers resistance against antibiotic compounds, for instance, multidrug-resistance Mycobacterium belongs to the actinobacteria phylum. Thus, the presence of pharmaceuticals in MBR could lead to the prevalence of actinobacteria. However, explaining the difference in the relative abundance of actinobacteria at different SRT is challenging. The increase in the relative abundance of phylum planctomycetes in phase 2 (6.29%) and phase 3 (4.79) and reduction in the relative abundance of Verrucomicrobia with the decrease in SRT indicates that the change in SRT of the MBR greatly influences the distribution of phylum in MBR. The decrease in SRT (from 100 to 45 days) increases the food to micro-organism ratio (F/M), which might lead to the proliferation of actinobacteria and planctomycetes, however, the discharge of a large amount of sludge at shorter SRT (15 days) did not provide adequate time to members of theses phylum (actinobacteria, planctomycetes) to propagate and the phylum with high growth rate (such as proteobacteria) became abundant.
Distribution of bacterial class at varying SRT
The study on phylum distribution at different SRT reveals a shift in bacterial community abundance with the change in operating condition, thus a higher taxonomic resolution could provide a clearer picture of interaction and correlation of bacterial community with the operating condition. The identified bacterial classes, i.e., nineteen classes were present in all the samples, however, their distribution varied greatly in varied phases (Fig. 4). Beta-proteobacteria were the abundant class in phase 1 and phase 3 with a relative abundance of 35. 3% and 36.8% whereas in phase 2, Alpha-proteobacteria were dominant. The relative abundance of Alpha-proteobacteria at 100, 45, and 15 days SRT were 5.8%, 41.23%, and 31.13%, respectively (Fig. 4). The beta-proteobacteria was reported to degrade polyaromatic hydrocarbons, surfactants, pharmaceuticals, and other emerging contaminants [27, 28]. Kara Murdoch et al.  demonstrated that the addition of chemical surfactant resulted in increased abundance of Beta-proteobacteria and they have the tendency to adapt to the sudden change in the environment. Thus, the dominance of Beta-proteobacteria in MBR treating pharmaceutical wastewater might be due to its inherent ability to survive in stress conditions. The high abundance of proteobacteria phylum in MBR is attributed to the increased abundance of Beta-proteobacteria and Alphaproteobacteria. The Alpha-proteobacteria consist of a large fraction of pathogens and symbionts and demonstrated to tend to remove non-functional genes from their genome . A study showed that the Alpha-proteobacteria and Gamma-proteobacteria carry 36% of antibiotic-resistance genes in the study of 16 metagenomes . Although in the present study, the antibiotic is not utilized, the inherent properties of Alphaproteobacteria to dispense non-functional genes and presence of antibiotic-resistance genes indicate that Alpha-proteobacteria could lead to the evolution of some mechanism (such as efflux pump, horizontal gene transfer, or drug resistance by altering drug or drug target) to proliferate in presence of pharmaceuticals in MBR. During the analysis of bacterial class in MBR at varying SRT, it was found that the reduction in SRT (from 100 to 45 days) increased proliferation of bacterial class reported to possess antibiotic-resistance mechanism, however, a further decrease in SRT, decreases the abundance of these bacterial classes. For instance, the Actinobacteria whose relative abundance was 0.90% in phase 1, showed an increase in relative abundance in phase 2, i.e., 7.47% but further it reduced from 7.4 to 3.30% at SRT 15 days in phase 3. Other than Actinobacteria, Gamma-proteobacteria, Cytophagia, and Clostridia showed a similar trend in increase and decrease in relative abundance at 45 days and 15 days SRTs (Fig. 4). These fluctuations in the relative abundance of bacterial class at varying SRT are possible due to the increased F/M ratio in MBR (high pharmaceutical concentration) at SRT of 45 days, which increase the proliferation of bacteria or pathogen having mechanism to fight against pharmaceutical contamination, However, the members of these bacterial class are slow-growing microbes which resulted in a decrease in relative abundance due to further decrease in SRT.
Distribution of bacterial genus at varying SRT
During the analysis of bacterial class, a shift was observed within the bacterial community of MBR in phase 1 and phase 2. The study on deeper taxonomic rank, i.e. genus was performed to provide insight into the MBR microbiome (Fig. 5). The analysis of genus-level distribution reveals that among the sixty-one dominant genus, only thirty-eight genera were shared in all the phases and eleven genera were unique to their corresponding phase. For instance, the genus Caldimonas, Aggregicoccus, Mangrovibacterium, Mucilaginibacter, and Piscinbacter were present only in Phase 1. The genus Asticcacaulis, Aureimonas, Albidiferax, Bdellovibrio, and Blastopirellula were confined only to phase 2 and the genus Alicyclobacillus was observed in phase 3. Apart from these, there are several genera, namely Aminobacter, Anaerorhabdus, Bacillus, Beijerinckia, Blastocatella, Campylobacter, Carnobacterium, Gimesia, Planctomicrobium, and Rubinisphaera, were shared in phase 2 and phase 3 but were absent in phase 1. Along with the difference in the distribution of genera, the relative abundance of bacterial genus varied greatly. For example, the dominant genus Hydrogenophaga having a relative abundance of 32.43% at SRT of 15 days constitute only 0.14 and 0.0174% at SRT of 45 and 100 days, respectively. Moreover, the abundant genus of phase 1, Caldimonas which accounts for the relative abundance of 21.1% was only identified in phase 1 and the most abundant genus of phase 2 Acetoanaerobium (relative abundance of 19.92%) were less than 0.1% in phase 1 and phase 3. The genus Caldimonas, Sulfurospirillum, Ferruginibacter, Haliangium, and Mucilaginibacter were most abundant in phase 1 having a relative abundance of 21.1, 12.7, 9.71, 5.6, and 5.4%, respectively. Whereas, in phase 2, Acetoanaerobium (19.92%), Acinetobacter (14.51%), Acidaminococcus (14.49%), Aciditerrimonas (9.69%) and Acidovorax (7.21%) were dominant. Likewise, in phase 3, Hydrogenophaga, Pseudorhodobacter, Gemmobacter, Rhodobacter, and Ferruginibacter having a relative abundance of 32.43, 12.311, 10.58, 5.46, and 4.6%, respectively, were abundant genera. The dissimilarities between genus profile at SRT of 100, 45, and 10 days unveil that the SRT is the important factor that decides the abundance and dominance of bacterial community in MBR.
MBR performance and bacterial community shift
The abundance of Alpha-, Beta-, and Gamma-proteobacteria, which were reported as an efficient degrader of organic matter and known to secret extracellular enzyme , explained the high removal of COD in the MBR. The increase in the dominance of glycogen accumulating organisms (GAO) belongs to Alphaproteobacteria in phase 2 and phase 3 probably decreases the orthophosphate removal observed at lower SRT of 45 and 15 days. Meyer et al. , identified the glycogen accumulating phenotypes in Alpha-proteobacteria and confirmed the deterioration of phosphorus removal in wastewater treatment plants due to the increased abundance of GAO which developed a competition for phosphate accumulating organisms (such as Actinobacteria) for survival and ultimately limits its growth and proliferation. The degradation of ammoniacal nitrogen in MBR decreases slightly with the decrease in SRT, which in general consistent with other related reported studies [3, 19, 33]. Wang et al.  studied the impact of SRT on salt accumulation in osmotic membrane bioreactor treating synthetic wastewater. The study revealed that lower SRT led to the accumulation of salt in the bioreactor which caused a high energy demand for osmotic adaptation of microbiome; thus, the removal efficiency of organic matter decreased. However, in the present study, the salt accumulation was not possible as an ultrafiltration membrane having a pore size of 0.04 µm was used, which did not provide retention of salt in MBR. Thus, the decrease in biomass from 15 (at SRT 100 days) to 6.7 g L−1 (at SRT of 15 days) affects the removal of ammoniacal nitrogen in MBR.
The performance of the ultrafiltration membrane was assessed by monitoring the trans-membrane pressure (TMP) of the membrane unit. The maximum TMP value prescribed by the manufacturer was 55 kPa. However, to increase the durability of the membrane, the maximum TMP during the operation was 40 kPa. Once the TMP was raised to 40 kPa, the membrane was washed with NaClO and citric acid to remove clogging. During the SRT of 100 days, the TMP reaches 40 kPa after the 30 days of operation. The decrease in SRT from 100 to 45 days decreased the clogging of MBR and the TMP increased to its maximum value after 40 days of operation. However, at shorter SRT (15 days), the clogging of the membrane increased and clogged after 20 days of operation. These findings are in agreement with the observation of Ouyang and Junxin . The increase in membrane fouling at a shorter SRT of 15 days was probably due to the presence of EPS and biofilm-forming microbes such as Hydrogenophaga (32.43%). Inaba et al.  reported a high abundance of Hydrogenophaga in the biofilm of MBR. The inherent characteristics of bacterium Hydrogenophaga to secrete polymeric substances might result in the accumulation of extracellular proteins and polysaccharides, which resulted in increased fouling of the membrane. Moreover, the study revealed a high abundance of Beta-proteobacteria in biofilm . The present finding indicates that the high abundance of Beta-proteobacteria at SRT 100 days and SRT 15 days (Fig. 4) caused a rapid clogging of the membrane. Since the removal of targeted pharmaceuticals was similar at varying SRT, the best SRT for the efficient treatment of pharmaceutical wastewater having similar wastewater characteristics could be 45 days. As discussed earlier, the clogging of the membrane can be reduced by optimizing the operating conditions of the MBR, such as SRT and HRT (Fig. 5).
Effect of SRT variation on bacterial community
The SRT variation significantly influences the bacterial community structure. The shift in dominance and relative abundance of bacterial community from one to another SRT was distinct. For instance, the phylum Armatimonadetes and Candidate were observed only at SRT 15 days. The prevalence of phyla Armatimonadetes was documented in the microbiome of wastewater treatment treating pharmaceutical and personal care products (PPCPS) [36, 37]. Kim et al.  investigated the bacterial community structure of biofilm reactor treating PPCPs using inocula obtained from a different source. The study revealed that inocula which were derived from sediments of the river were more efficient in the removal of pharmaceuticals (ibuprofen, diclofenac, and gemfibrozil). One of the major bacterial phyla of sediment derived was Armatimonadetes. These findings and the presence of Armatimonadetes in MBR indicated that Armatimonadetes could be responsible for pharmaceutical removal. However, the phyla Armatimonadetes were recently defined and information on the Armatimonadetes genome was not available yet. The relative abundance of phyla Verrucomicrobia decrease successively with the decrease in SRT, i.e., from 3.83 to 0.13%, and finally to 0.09% at SRT of 100, 45, and 15 days, respectively, supported the assumption that members of Verrucomicrobia adapt k-selection strategy. The k-selection evolutionary strategy involves slow-growing organisms and k-selected species occupy a stable environment . The presence of phylum Deinococcus-Thermus, and Ignavibacteriae in phase 2 and phase 3 indicates that the lower SRT provides the feast condition within the reactor, which resulted in the proliferation of these phyla.
Bacterial community associated with pharmaceuticals removal
Despite these fluctuations in the bacterial community of MBR, the removal of ibuprofen and estradiol was constant at varying SRT, i.e. > 90%. Fortunato et al.  performed the biodegradation study of ibuprofen with the indigenous bacterial community. The study revealed that the Comamonas and Bacillus sp. were the dominant genus in the fixed bed continuous reactor and able to degrade 95.9% of ibuprofen. The presence of Bacillus and Comamonas in MBR in all the treatment conditions (Phase 1, 2 and3) suggested that the ibuprofen was degraded in the MBR. The biodegradation of estradiol involves the transformation of estradiol to estrone followed by its mineralization . A study reported that the concentration of estradiol causes changes in the abundance of the degrading bacterial community of wastewater treatment plants . The micro-autoradiography-fluorescence in situ hybridization demonstrates that the high estradiol concentration resulted in the proliferation of Beta-proteobacteria and the abundance of Alphaproteobacteria increased with a decrease in concentration (in µg L−1) . As the SRT reduces, the amount of excess sludge withdrawn from the reactor increases, which implies that estradiol residues absorbed in the sludge also reduces (discharge with sludge). Thus, the low concentration of estradiol in MBR at lower SRT might lead to the proliferation of actinobacteria at lower SRT. It was evident that a decrease in SRT, increased the relative abundance of Alphaproteobacteria from 5.8% at 100 days SRT to 31% at 15 days SRT. The negligible removal of CBZ even at a longer SRT of 100 days in MBR was in line with the study reported by Gonzalez-Perez et al. . A study on respirometric analysis of activated sludge spike with CBZ revealed that the chemical stability and hydrophilic characteristics prevent its bioavailability to micro-organisms. Moreover, the addition of CBZ caused a drop in oxygen uptake rate (OUR), however, after few hours the OUR was recovered due to the metabolic adaptation of the micro-organisms . The removal of venlafaxine was limited in MBR (< 20%). The comparative study on removal of venlafaxine in aerobic and anaerobic sequence batch reactor revealed that anaerobic treatment was efficient in degradation of > 60% of venlafaxine via demethylation process .
In this study, it was observed that a change in SRT significantly influenced the relative abundance of bacterial communities at the genus level. Moreover, the presence of bacteria involves in the degradation of heterocyclic aromatic hydrocarbon, phenols, polychlorinated biphenyls, and xenobiotics were observed in MBR. For instance, the presence of bacteria Dyadobacter and Beijerinckia reported to degrade aromatic hydrocarbon, was detected in MBR at varying SRT. Kim et al.,  suggested that the presence of these bacterial genera in biomass involved in the treatment of pharmaceutical-containing wastewater possibly have some role in the degradation of pharmaceutical compounds. The bacterium Dyadobacter belongs to the phylum Bacteroidetes, members of whose reported to possess antibiotic-resistance genes. The studies assessing the impact of SRT on the bacteria community of MBR treating synthetic wastewater reported no significant effect of SRT on the bacterial community [43, 44]. However, in the present study, a substantial change in the bacterial community of MBR at varying SRT pointed toward the fact that the pharmaceutical presence affected the dynamics of the bacterial community. This investigation is in agreement with the previous study ; however, variation in HRT did not affect the bacterial community composition. In the present study, the change in the composition of the bacterial community along with the change in SRT was observed, which suggests that the concentration of pharmaceutical absorbed on the sludge and the duration of exposed pharmaceutical to MBR biomass affects the bacterial community dynamics.
The study highlights the effect of pharmaceuticals on bacterial community composition, however, other members of the wastewater microbiome (micro-eukaryotes and archaea community) were not included in the present study. To understand the insights of the role of the microbial community in the removal of pharmaceuticals, and the biodegradation pathway of pharmaceuticals in micro-organisms, studies on the microbiome of wastewater treatment plants (WWTP) treating pharmaceuticals containing wastewater (such as hospital wastewater and/or pharmaceutical industry wastewater) are required. Moreover, the presence of pharmaceuticals could also result in the proliferation of ARB in WWTP. Thus, detailed research is required for the detection and inactivation of ARGs and ARBs and the proper disposal of the sludge. To prevent the emergence of ARGs and ARBs, an efficient system with less sludge production and strict regulation is needed.
The study assessed the impact of SRT on the bacterial community of MBR treating synthetic hospital wastewater. The removal of pharmaceutical was constant at varying SRT, i.e., > 90% for IBF and EE2, < 20% for VNF. The MBR was not able to remove CBZ. The presence of pharmaceuticals and change in SRT alter the dynamics of the bacterial community and a significant shift in the bacterial community structure of MBR biomass was noticed. The presence and abundance of biofilm-producing and antibiotic-resistance bacteria reported, such as Hydrogenophaga, Dyadobacter, and Beijerinckia, in different phases of the MBR indicate that both pharmaceutical presence and its concentration alter the bacterial community composition. The study suggested that the prolonged exposure to pharmaceuticals is responsible for the proliferation of bacteria which can degrade these pollutants. Detailed investigations on the role of the microbiome (including micro-eukaryotes) on pharmaceutical removal are recommended.
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The authors thank the Natural Sciences and Engineering Research Council of Canada, Canada (NSERC-STPGP-479160 Strategic grant, Premier Tech Ltée, Canada Research Chair) for financial support.
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The corresponding author declares no conflict of interest whatsoever. Moreover, all the content, tables, and figures including drawings, charts, photographs, etc. are original work in the submitted manuscript.
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Tiwari, B., Sellamuthu, B., Piché-Choquette, S. et al. Dynamics of bacterial community at varying sludge retention time within membrane bioreactor treating synthetic hospital wastewater. Syst Microbiol and Biomanuf 1, 471–482 (2021). https://doi.org/10.1007/s43393-021-00034-y
- Membrane bioreactor
- Sludge retention time
- Bacterial community