Extracellular Organic Matter from Micrococcus Luteus Containing Resuscitation-Promoting Factor in Sequencing Batch Reactor for Effective Nutrient and Phenol Removal

Science of the Total Environment 727 (2020) 138627

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Science of the Total Environment

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Extracellular organic matter from Micrococcus luteus containing resuscitation-promoting factor in sequencing batch reactor for effective nutrient and phenol removal

Chunna Yu a,1, Yindong Liu b,c,1, Yangyang Jia b, Xiaomei Su b,d, Lian Lu b, Linxian Ding d, Chaofeng Shen b,⁎ a College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China b Department of Environmental Engineering, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China c Environmental Protection Bureau of Zhejiang Province, China d College of Geography and Environmental Science, Zhejiang Normal University, Jinhua 321004, China

HIGHLIGHTS GRAPHICAL ABSTRACT

• Effect of SRpf from Micrococcus luteus on biological nutrient and phenol removal was evaluated in lab-scale SBR. Phenol • SRpf enhanced the phosphorus, nitrogen and phenol removal. Reactor 1 Reactor 2 Reactor 3 • The phyla of Proteobacteria and Inactivated SRpf SRpf Control Group SRpf: Culture supernatant containing Actinobacteria related to nutrient and resuscitation-promoting factor from Micrococcus luteus phenol removal were abundant with SRpf addition. Nitrogen Phosphorous

article info abstract

Article history: Culture supernatant containing resuscitation-promoting factor (SRpf) from Micrococcus luteus was added to the Received 11 December 2019 sequencing batch reactor (SBR) for effective treatment of phenol-containing wastewater. SRpf acclimation signif- Received in revised form 8 April 2020 icantly improved combined removal of phenol and nutrients. Moreover, the Illumina high-throughput sequenc- Accepted 9 April 2020 ing analysis revealed that the SRpf boosted bacteria diversity, which enhanced the stability of the system under Available online 13 April 2020 phenol stress. Addition of SRpf increased the abundances of Actinobacteria and Proteobacteria phyla which are in- fi Editor: Fang Wang volved in nutrient and phenol removal. Speci cally, SRpf promoted NitrosomonasandNitrospira which partici- pate in nitrification, family Comamonadaceae,generaDechloromonas and Pseudomonas involved in Keywords: denitrification, and Acinetobacter, Pseudomonas and Rhodocyclus which remove phosphorus elements. Moreover, Sequencing batch reactor the abundances of Bacillus and Klebsiella responsible for phenol removal as well as Pseudomonas and Activated sludge Acinetobacter were significantly increased after SRpf acclimation. These results show that SBR combined with Bacterial community SRpf acclimation provide optimal nutrient and phenol removal. Viable but non-culturable state © 2018 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Department of Environmental Engineering, Zhejiang University, Yuhangtang Road 866, Hangzhou 310058, China. E-mail address: [email protected] (C. Shen). 1 Co-ﬁrst author.

1. Introduction 2.2. Lab-scale sequencing batch reactors (SBRs)

Several restrictions have been proposed to reduce the environmen- Activated sludge collected from the secondary sedimentation tank in tal toxicity due to wastewater from industries. For this reason, wastewa- Qi Ge Domestic Wastewater Treatment Plant (Hangzhou, China) was ter treatment plants (WWTPs) face enormous challenge given that the employed as the inoculum for SBRs. Three parallel SBRs (2.5 L working currently used methods are not sufficiently effective in removing nutri- volume each) were utilized in this work (Liu et al., 2016). Each bioreac- ent and toxic organic compounds from wastewater (Wang et al., 2015; tor had an 8 h cycle with the following sequence: 10 min of feed of Winkler et al., 2011). For removal processes of nutrient and toxic or- 1.25 L influent synthetic wastewater, 50 min of anaerobic phase, 3.5 h ganic compounds, improving activity of microbial community is a of aerobic phase, 2.5 h of anoxic phase, 35 min of sedimentation, and promising way to overcome the low efficiency of biological process, be- 25 min of extracting 1.25 L of the supernatant. The primary components cause lots of functional bacteria are at the state of low metabolism or of synthetic wastewater used in the test are shown in Table A. 1 (Coats even dormancy state, known as the “viable but non-culturable” et al., 2011). The wastewater had a chemical oxygen demand (COD) of (VBNC) state (Epstein, 2013; Su et al., 2013a). about 620 mg·L−1, total nitrogen (TN) of 54 mg·L−1, ammonia nitrogen + −1 −1 A bacterial cytokine named resuscitation-promoting factor (Rpf), (NH4 -N) of 48 mg·L , total phosphorous (TP) of 16 mg·L ,andpHof which was firstly identified in Micrococcus luteus, resuscitates VBNC 6.9–7.3. bacteria (Kaprelyants and Kell, 1993; Mukamolova et al., 1998). So The three reactors were allowed to for run steadily for 30 d before far, Rpf and Rpf-like cytokine have been identified in several gram- addition of phenol and SRpf acclimation. The concentrations of TN and positive and gram-negative bacteria species (Senoh et al., 2012). TP in the effluent reduced to a stable level of 6.5 mg·L−1 and Rpf has been reported to resuscitate and stimulate numerous 2.5 mg·L−1, respectively. Phenol was then added to the influent with in- gram-positive and gram-negative bacteria (Ding and Yokota, 2010: creasing concentrations as follows (Fig. A. 1): 20 mg·L−1 in phase 1 Nikitushkin et al., 2011). Although the underlying mechanism of (1–7d),50mg·L−1 in phase 2 (8–13 d), 100 mg·L−1 in phase 3 Rpf remains unclear, some studies have reported that it may influ- (14–20 d) and 150 mg·L−1 in phase 4 (21–30 d). The SBRs was run ence the environmental effects of VBNC bacteria and remove pollut- under 25 ± 0.5 °C for 30 d. Artificial sludge discharge was carried out ants. Recently, the purified Rpf from M. luteus was shown to improve to keep the biomass at the similar level during each operation cycle in pollutant degradation capacity under high salinity conditions by re- the experiment. We continuously measured the mixed liquor volatile suscitating and stimulating the potential pollutant-degrading bacte- suspended solids (MLVSS) of the system during the experiment, and ria (Su et al., 2018a; Su et al., 2018b; Su et al., 2019a). Moreover, the concentration of MLVSS in the three reactors was always around other Rpf-responsive bacterial populations in phyla Proteobacteria, 4100 mg·L−1 (Tables A. 2 and A. 3). Therefore, the biomass was kept Bacteroidetes,andActinobacteria have been well documented (Su steady in R1, R2 and R3. et al., 2019b: Su et al., 2019a). Thus, we infer that Rpf can be used a supplement to obtain high-efficient pollutant-degrading strains 2.3. Acclimation of activated sludge using SRpf and boost the biodegradation process in wastewater treatment. However, the activity of pure Rpf protein from M. luteus culture su- The acclimation of sludge by SRpf was optimized as previously de- pernatant or recombinant Rpf decreased beyond one week at 4 °C scribed (Liu et al., 2016). Briefly, during the sedimentation phase in (Su et al., 2013b). Besides, several other unknown but functionally each cycle, 100 mL of the condensed sludge were obtained from the similar proteins are present in supernatants obtained from treatment reactor R2, acclimated using SRpf (10%, v·v−1) in a rotary M. luteus (Mukamolova et al., 2006). Therefore, resuscitating and shaker (25 °C, 150 rpm), and returned to the reactor before the start stimulating the bacteria using the culture supernatant from of the next cycle. The inactivated SRpf (sterilized at 121 °C for 15 min) M. luteus containing Rpf (SRpf) rather than purified Rpf protein was used to acclimate the sludge for the control reactor R1. The syn- seems to be a more effective approach (Su et al., 2013a). However, thetic wastewater with the same concentrations of COD and nutrient the relevant research is very limited. A recent study showed that as SRpf were then utilized to treat the sludge for the blank reactor R3 employing SRpf to acclimatize activated sludge in a lab-scale se- and which was then returned to R1 and R3, respectively. quencing batch reactor (SBR), accelerates the start-up process and significantly enhances the removal of phosphorus and nitrogen. 2.4. Chemical analysis Illumina high-throughput sequencing analysis showed that SRpf im- + − − 3− proves the diversity and activity of the microbial community by re- The concentrations of COD, NH4 -N, NO3 -N, NO2 -N, and PO4 -P in moving nutrients (Liu et al., 2016). However, whether SRpf can the influent and effluent were monitored as per the APHA standard improve treatment of wastewater containing toxic organic com- methods (APHA, 2005). Phenol measurement was performed using an pounds in SBR, remains unknown. HPLC with a UV–Vis detector (LC-2010A HT, Shimadzu) while other pa- In the present study, we compared the removal efficiencies of nutri- rameters were as described previously (Liu et al., 2016). ent and phenol in three different SBR reactors with/without SRpf and explored the bacterial community compositions of the sludge samples. 2.5. Bacterial community analysis We aimed to determine whether SRpf can be used as supplement to treat toxic organic wastewater and enhance functional bacteria, which As shown in Fig. A. 1, the experiment was divided into four phases could facilitate nutrient and phenol removal. with influent phenol concentration of 20 mg·L−1 (phase 1), 50 mg·L−1 (phase 2), 100 mg·L−1 (phase 3), and 150 mg·L−1 (phase 4), respectively. At each phase, after the reactor had been running 2. Materials and methods steadily for about 3 cycles, activated sludge samples for DNA extraction of reactor 1, reactor 2, and reactor 3 were collected in the end of certain 2.1. Preparation of SRpf from M. luteus cycle. Twelve samples were collected from Reactor 1 (R1-1, R1-2, R1-3 and R1-4), R2 (R2-1, R2-2, R2-3 and R2-4) and R3 (R3-1, R3-2, R3-3 SRpf was prepared as described previously (Su et al., 2013b). and R3-4), and the sample Rx-y represented the sample collected M. luteus (AJ409096.1) were inoculated in minimal lactate medium from the x reactor in operation phase y. Next, DNA was isolated from (LMM), centrifuged, and filtered to obtain a cell-free supernatant con- the sludge using the Fast DNA SPIN Kit for Soil (Bio101 Inc., USA). PCR taining Rpf (SRpf) (Kaprelyants and Kell, 1993). After that, the SRpf was performed using the 319F/806R primer set, which amplified the was stored at −20 °C for use. V3-V4 region of the 16S rRNA gene of the DNA extracts. The samples C. Yu et al. / Science of the Total Environment 727 (2020) 138627 3 were sequenced on an Illumina MiSeq platform (Guheinfo, Hangzhou, 3. Results and discussion China). All raw reads have been deposited into the NCBI Sequence Read Archive (Accession Number: SRR1948176). We employed the 3.1. Effects of SRpf on long-term performance of SBR under phenol stress Quantitative Insights Into Microbial Ecology (QIIME) software package to analyze the sequences which were assigned to operational taxonomic In the continuous operation, we aimed to examine whether R2 with units (OTUs) at 97% similarity and custom Perl scripts to investigate SRpf treatment enhances nutrient removal under phenol stress com- alpha and beta diversity (Wang et al., 2007). pared to the inactive SRpf control R1 or blank control R3 in the long- term. 2.6. Statistical analysis Notably, the efficiency of phosphorous removal was significantly influenced by the phenol load shock (Fig. 1a). At the beginning of phase 1 −1 3− Statistical analysis was performed using the Data Processing System containing 20 mg·L phenol, the concentration of PO4 -P in all the (DPS) software (version 11.0), using the least significant difference three bioreactors effluent was about 8 mg/L, which was higher than be- (LSD) test for each pair of data sets. We considered the significance of fore phenol was added (2.5 mg·L−1). After that, phosphorous concen- the SRpf acclimation effects as p b 0.05. Chao1 metric was used to esti- tration in the effluents of the three SBRs was gradually reduced to mate the species richness, while we utilized the Shannon index to assess near levels before phenol loading. Of note, there was no significant dif- bacterial diversity. ference in phosphorous concentration between R2 and R1 or R3.

3− −1 Fig. 1. Variation of efﬂuent concentrations of PO4 -P (a) and total nitrogen (b) in reactor R1, R2 and R3 at four phases with inﬂuent phenol concentration of 20 mg·L (phase 1), 50 mg·L−1 (phase 2), 100 mg·L−1 (phase 3), and 150 mg·L−1 (phase 4), respectively. 4 C. Yu et al. / Science of the Total Environment 727 (2020) 138627

However, in phase 2 with 50 mg·L−1 phenol adding, the average P re- process of denitrifying P removal under phenol stress, maybe through moval rate in R2 (56.1%) was significantly higher (p b 0.05) than in R1 promoting specific functional bacteria. (48.4%) or R3 (46.6%). The activated sludge with SRpf acclimation began to show stronger toxicity resistance-capacity with increasing 3.2.2. Nitrogen removal −1 + − phenol levels. In phase 3, containing 100 mg·L phenol loading, the in- A typical profile of different nitrogen species (NH4 -N, NO3 -N, and − hibition of phosphorous removal was reduced to a smaller extent. At the NO2 -N) is shown in Fig. 2b at a steady state in the three reactors with beginning of the phase (14 d), the average P removal rates were 38.5%, phenol adding. Overall, the efficiency of nitrogen removal was signifi- 48.8%, and 40.4% in R1, R2, and R3, respectively. This shows that the cantly higher in R2 with SRpf acclimation compared to R1 and R3 in a phenol degraders were enriched in the three bioreactors. Bioreactor typical cycle. + R2 performed much better in recovery from high-level phenol shock At the end of the aerobic stage, the removed amount of NH4 -N in R2 for P removal. At the end of phase 3 (20 d), the P removal rate in R2 (20.44 mg·L−1) was significantly higher than in R1 (18.45 mg·L−1)and (73.6%) was significantly higher (p b 0.05) than in R1 (58.1%) or R3 R3 (16.75 mg·L−1)(p b 0.05) (Table A. 3). Correspondingly, the nitrifi- (60.0%). When the phenol reached high (150 mg·L−1) in phase 4, the cation rate in R2 (0.62 mg·gVSS−1·h−1) was also significantly higher phosphorous removal capacity in the three systems, declined immedi- than in R1 (0.41 mg·gVSS−1·h−1) or R3 (0.36 mg·gVSS−1·h−1) − ately. However, R2 still showed higher efficiency all through phase 4. (p b 0.05). The R2 NO3 -N concentration, which was significantly higher On the last day of the phase (30 d) with 150 mg·L−1 phenol stress, compared to R1 or R3 increased through the whole aerobic stage 3− the removal rate of PO4 -P in R2 was above 60%, while it was lower (p b 0.05). The start-up of nitrification was delayed in R1 or R3 through than 50% in both R1 and R3. Evidence shows that the removal rate of phenol inhibition, while no delay was reported in R2. We monitored the − phosphorous declines by 30%–40% under phenol stress in SBR (Uygur accumulation of NO2 -N in all the three reactors. However, the concen- − and Kargi, 2004). This is consistent with our results in R1 and R3, tration of NO2 -N in R2 was significantly higher than in R1 or R3 at the while in R2, there was relatively high efficiency of phosphorous removal earlier period (p b 0.05), while its level in R2 reached the lowest in even with hash shock of phenol. Therefore, SRpf acclimation of sludge the three SBRs at the end of the aerobic stage (p b 0.05). Phenol is promotes a high rate of phosphorous removal in the SBR system toxic to nitrite-oxidizing bacteria (NOB); therefore, it leads to the accu- − under phenol stress. mulation of NO2 -N (Li et al., 2010; Neufeld et al., 1986). However, NOB The efficiency of nitrogen removal was also considerably influenced with SRpf acclimation had more stress resistance to phenol for nitrite following the addition of phenol (Fig. 1b). Besides, the variation trends oxidation. At the anoxic stage, no evident denitrification was reported of TN concentrations in the effluents of the three SBRs were similar to in R1 or R3, while the denitrification rate was high (0.35 mg·gVSS−1·- phosphorous. In phase 1, with 20 mg·L−1 phenol adding, SRpf acclima- h−1) in R2 (Table A. 4). Therefore, denitrifying bacteria with SRpf accli- tion did not show any significant enhancement of phenol tolerance in mation also had more resistance to phenol stress. R2. However, in phase 2 (50 mg·L−1 phenol), phase 3 (100 mg·L−1 phenol), and phase 4 (150 mg·L−1 phenol), the TN concentration in 3.2.3. Nitrification inhibition by phenol R2 effluent was significantly lower than in R1 or R3. At the end of As shown in Fig. 3a and compared with the previous results without phase 4, the TN removal rate in R2 was higher than 60%, while in R1 phenol stress (Liu et al., 2016), the inhibition of nitrification by phenol or R3, it was lower at 40%–50%. was evident, and the results are consistent with the first-order kinetic Research evidence indicates that SRpf facilitates nutrient removal in equation of single-molecule inhibition. The formula and detailed kinet- a 130d operation of SBR (Liu et al., 2016). Our results showed that ics parameters can be referred to in the supporting information. The in- sludge with SRpf acclimation has more resistance to phenol stress for hibition rate of nitrification in R2 under different phenol loading nutrient removal compared to the controls in the long-term. concentration was significantly (p b 0.05) lower compared to R1 or R3 (Table A. 4). On the contrary, the inhibition constant from model fitting increased with reducing inhibition. In R2, the inhibition constant was 3.2. Effects of SRpf on a typical cycle of SBRs − 333.47 mg·L 1 (Fig. 3a), which was significantly higher (p b 0.05) that in R1 (98.77 mg·L−1) and R3 (94.57 mg·L−1). Altogether, these re- We monitored a typical cycle (phase 3, 100 mg·L−1 phenol adding) sults indicated that there was a much lower nitrification inhibition in R2 to assess whether R2 with SRpf acclimation enhances nutrient and phe- from phenol stress than in R1 or R3. nol removal in the short-term. The inhibition constant of nitrification by phenol varies considerably among different studies based on the different activated sludge, running 3.2.1. Phosphorus removal condition, among other factors. In some studies, about 21 mg·L−1 inhi- In each stage (anaerobic, aerobic, or anoxic stage) of the operation, bition constant for enriched nitrification sludge, has been reported although all the three reactors showed a reduction in the rate of phos- (Blum and Speece, 1991; Xu et al., 2005), while the constant is phorus (P) removal after phenol adding, comparatively better efficiency 52.87 mg·L−1 in the short-cut nitrification sludge (Lu et al., 2012). In was achieved in R2 (Fig. 2a; Table A. 2). At the anaerobic stage, the this study, the activated sludge was not enriched and had abundant het- amount of P released (5.45 mg·L−1), or P release rate erotrophic bacteria. Therefore, the system had more detoxification abil- (1.77 mg·gVSS−1·h−1) in R2, was significantly higher than in R1 or ity to phenol; hence, had higher nitrification inhibition constant. In R2 R3 (p b 0.05). In the aerobic stage, the amount of P uptake in R2 was with SRpf acclimation, the inhibition constant was remarkably high, in- 13.12 mg·L−1，while in R1 and R3, it was only 9.52 mg·L−1 and dicating that SRpf enhanced the stability and tolerance of the microbial 9.31 mg·L−1, respectively. Correspondingly, the P uptake rate in R2 community to phenol stress. was 0.91 mg·gVSS−1·h−1，while the rates in R1 and R3 were only 0.65 mg·gVSS−1·h−1 and 0.64 mg·gVSS−1·h−1, respectively. In the an- 3.2.4. Phenol removal and its priority over nitrification oxic stage, P uptakes were also observed across the three SBRs. The bio- Regarding phenol removal in a typical cycle (Fig. 3b), R2 showed a mass in R2 assimilated significantly more phosphorus much higher rate of phenol degradation (21.7 mg·gVSS−1·h−1) com- (3.73 × 10−2 mg·gVSS−1·h−1) than in R1 (0.40 × 10−2 mg·gVSS−1·- pared to R1 (8.53 mg·gVSS−1·h−1) or R3 (9.22 mg·gVSS−1·h−1). Phe- h−1), or R3 (0.81 × 10−2 mg·gVSS−1·h−1)(p b 0.05). The secondary nol was removed entirely in 1.5 h for R2 and 3.5 h for R1 or R3 in the phosphorous release is negligible at the post anoxic stage as long as ni- aerobic stage. Therefore, R2 with SRpf acclimation enhances phenol re- trate/nitrite remains available (Winkler et al., 2011; Carvalho et al., moval in the short-term. 2007), which was consistent with our results as well as previous find- As shown in Fig. 3, there was no removal of ammonium nitrogen as ings (Liu et al., 2016). Therefore, SRpf significantly accelerated the well as phenol in the anaerobic stage. However, in the first 1.5 h of the C. Yu et al. / Science of the Total Environment 727 (2020) 138627 5

Fig. 2. Variations of phosphorous (a) and nitrogen (b) during one typical cycle (phase 3, 100 mg·L−1 phenol adding) in reactor R1, R2 and R3.

+ + aerobic stage, both NH4 -N and phenol simultaneously reduced in all the NH4 -N was relatively abundant. However, the nitrification rate in R2 + −1 three reactors. Although nitrification was suppressed by phenol degra- did not increase because of low NH4 -N (5 mg·L ) level. Similar to + dation in 1.5 h, the removal rate of NH4 -N was high (64.9%) in R2 com- our findings, research evidence shows that the activity of ammonia- + pared to R1 (35.3%) and R3 (37.7%). Phenol degradation occurs in the oxidizing bacteria is affected by the presence of both NH4 -N and phenol organics' removal stage as well as the nitrification stage (Lee and Park, (Liu et al., 2005). Therefore, the inhibition by toxic organic compounds 1998) consistent with our findings. After 2 h of the aerobic stage, the such as phenol in activated sludge should be alleviated to achieve opti- phenol in R1 and R3 was reduced to b40 mg·L−1, while the phenol in mal wastewater treatment effect. Notably, SRpf acclimation was shown R2 was entirely degraded. The nitrification rates increased evidently in to effectively alleviate phenol inhibition, enhancing the treatment ca- R1 and R3 as phenol stress was alleviated to a considerable extent and pacity of the SBR system. 6 C. Yu et al. / Science of the Total Environment 727 (2020) 138627

Table 1 Comparison of bacterial diversity estimation of 12 activated sludge samples at 3% dissim- ilarity from reactor R1, R2 and R3 with phenol adding in long-term operation.

Samples Reads OTUs Chao 1 richness estimation Shannon diversity

R1-1 41,821 2993 4978 6.19 R2-1 41,482 3118 5299 7.23 R3-1 46,473 2860 4320 6.07 R1-2 28,138 2298 4162 6.42 R2-2 34,601 2836 5289 7.72 R3-2 23,045 2134 3423 6.34 R1-3 35,492 3399 4639 7.38 R2-3 56,985 4452 6372 8.48 R3-3 36,614 3170 4186 7.73 R1-4 27,842 2749 4656 7.88 R2-4 27,683 3592 5313 8.65 R3-4 50,382 2891 4566 7.56

phase 4. However, the abundance of Proteobacteria in R2 was consis- tently the highest in all the three reactors across all the phases. More- over, the average abundance in R2 (33.2%) was significantly higher (p b 0.05) than in R1 (24.9%) or R3 (26.1%). The abundance of Proteobacteria in the different bioreactors corresponded with the re- spective reactor's effluent quality. SRpf acclimation increased the abundance of Proteobacteria in the long-term. The subdominant group was Actinobacteria phylum (19.9%–27.4%) with the average abundance being R2 (26.6%) N R1 (22.8%) N R3 (22.0%) throughout all the phases consistent with previous findings (Liu et al., 2005). A similar trend was reported for the Nitrospirae, Proteobacteria,andActinobacteria phyla. Therefore, compared with R1 or R3, R2 had a higher abundance of Actinobacteria, Proteobacteria,andNitrospirae, which could be associ- ated with the effect of the SRpf protein (Su et al., 2013b). These main groups consisted of a significant number of polyphosphate accumulat- ing organisms (PAOs), nitrifiers, denitrifiers and phenol degraders, such as Accumulibacter, Dechloromonas, Nitrosomonas, Acinetobacter, Ba- cillus, Pseudomonas, among others. A variety of bacterial genera have been reported to response to SRpf, while almost all the SRpf has was just been found in gram-positive bacteria of the Actinobacteria phylum Fig. 3. Curves of phenol inhibition kinetics (a) and variations of phenol during one typical (Mukamolova et al., 2006; Nikitushkin et al., 2016). Therefore, SRpf − cycle (phase 3, 100 mg·L 1 phenol adding) (b) in reactor R1, R2 and R3. has the ability of resuscitate and enhance several cross-species bacteria. In this study, addition of SRpf not only increased the abundance of Actinobacteria but also the abundance of Proteobacteria and Nitrospirae compared with the control groups. It seemed that SRpf also had signifi- 3.3. Shifts in the bacterial community structure cant resuscitation and growth stimulating effects on bacteria that could not produce SRpf. We collected twelve sludge samples for bacterial community The population number of β-Proteobacteria was the highest among analysis from the three SBRs on typical dates of the four phases the Proteobacteria across all the samples, followed by γ-Proteobacteria. with increasing phenol addition (Fig. A. 1). As shown in Table 1, Research evidence indicates that β-Proteobacteria is always the most trimmed-high quality reads 23,045–56,985 were obtained after fil- abundant in activated sludge, and β-Proteobacteria and γ-Proteobacteria tering the low-quality reads for the 12 samples. The average opera- play a key role in nutrients removal. Furthermore, the averaging abun- tional taxonomic units (OTUs) adopted to estimate bacterial dance of β-Proteobacteria (18.5%) and γ-Proteobacteria (12.1%) in R2 species richness at a distance level of 3% was 3450 in R2 under SRpf were significantly higher (p b 0.05) than in R1 (13.9%, 7.7%) and R3 acclimation, while the average OTUs in R1 and R3 were 2860 and (14.2%, 8.6%). There were no significant differences in abundance in α- 2764, respectively. Chao 1 richness showed similar trends in these Proteobacteria or δ-Proteobacteria or ε-Proteobacteria among the three samples. The samples with SRpf acclimation had higher Shannon di- reactors and these three classes composed a relatively low proportion versity compared to samples from the inactivated SRpf or without in all the samples. In conclusion, even under phenol stress, SRpf in- SRpf reactors (p b 0.05). Previously, it was shown that SRpf acclima- creased the abundance of β-Proteobacteria and γ-Proteobacteria and en- tion significantly promotes bacterial diversity in SBR (Liu et al., hanced Proteobacteria in R2. These findings are consistent with the 2016). Similarly, in this study, even in the presence of phenol stress, previous study's results in which the wastewater had no toxic com- SRpf acclimation enhanced bacterial diversity. pound (Liu et al., 2016), indicating that β-Proteobacteria and γ- The distributions of the reads at the phyla and class level in Proteobacteria responded to SRpf. Proteobacteria are presented in Fig. A. 2. Proteobacteria was the major As shown in Table 2, within β-Proteobacteria, the predominant or- phylum constituting 22.1%–38.2% of total bacterial reads. Compared to ders were Rhodocyclales, Burkholderlales,andNitrosomonadales.The 33.9%–56.3% in the bioreactor without phenol stress (Liu et al., 2005), Rhodocyclales comprised of abundant bacteria responsible for nitrogen the abundance of Proteobacteria in this study was relatively lower. The and phosphorus removal, including Rhodocyclaceae, Dechloromonas, increase of phenol loading increased the abundance of Proteobacteria among others. The dominant family Comamonadaceae in Burkholderlales in all the three reactors (from phase 1 to 3) and gradually stabilized in is involved in denitrification (Etchebehere et al., 2001; Khan et al., C. Yu et al. / Science of the Total Environment 727 (2020) 138627 7

Table 2 responsible for phosphorus removal in R2 (3.3%) was significantly The relative abundance of the dominant orders of the phyla β-Proteobacteria and γ- higher (p b 0.05) than in R1 (2.3%) or R3 (2.4%). Moreover, the average Proteobacteria 12 activated sludge samples. abundance of Comamonadaceae (responsible for denitrification) in R2 Class level Order level Percentage/% (2.1%) was significantly higher (p b 0.05) compared to R1 (0.9%) or R3 R1-1 R2-1 R3-1 R1-2 R2-2 R3-2 (0.9%). Within the γ-Proteobacteria, the predominant orders were β-Proteobacteria Rhodocyclales 9.34 10.85 8.94 8.32 11.22 8.07 Burkholderlales 1.45 2.67 1.66 1.53 2.59 1.36 Pseudomonadales, Xanthomonadales,andLegionellales (Table 2). Nitrosomonadales 0.48 1.22 0.67 0.78 1.33 0.53 Pseudomonadales consisted of abundant Pseudomonas and Acinetobacter γ-Proteobacteria Pseudomonadales 2.03 3.35 2.98 2.78 5.17 2.92 genera responsible for nitrogen and phosphorus removal as well as phe- Xanthomonadales 2.13 3.21 1.55 1.97 3.14 1.43 nol degradation. The abundance of Pseudomonadales in R2 was signifi- Legionellales 0.15 0.18 0.17 0.21 0.22 0.15 cantly higher (p b 0.05) than in R1 and R3. Notably, Pseudomonadales Class level Order level Percentage/% was gradually enriched along with the gradient phenol adding, imply- ing it played a crucial role in phenol degradation. Moreover, SRpf accli- R1–3R2–3R3–3R1–4R2–4R3–4 mation enhanced the enrichment of phenol this degrader. β -Proteobacteria Rhodocyclales 9.16 11.85 9.24 9.25 12.23 10.06 To further assess changes in the bacterial community after addition Burkholderlales 1.69 2.97 1.66 1.73 3.88 1.89 Nitrosomonadales 0.71 1.25 0.77 0.74 1.17 0.81 of SRpf, we investigated the sequence distribution results of all samples γ-Proteobacteria Pseudomonadales 4.28 8.33 3.89 3.81 7.69 3.95 at the genus level using a heat map. The top 10 most abundant genera in Xanthomonadales 2.07 3.08 2.43 2.12 2.93 2.26 each sample (48 genera in 12 samples) were selected and compared Legionellales 0.24 0.28 0.25 0.2 0.19 0.22 with the abundance of other samples (Fig. 4). SRpf acclimation had a significant effect on the abundance of nitrifying bacteria, denitrifying bacteria, and PAOs under phenol stress. Nitrosomonas and Nitrospira 2002), while the primary family in Nitrosomonadales participates in consisted the two typical genera of the Ammonia-oxidizing bacteria nitrosation. Furthermore, under the gradient phenol stress, the abun- (AOB) and NOB, respectively. The average abundances of Nitrosomonas dances of these three orders in β-Proteobacteria, Rhodocyclales, and Nitrospira in R2 were significantly higher than compared to R1 or R3 Burkholderlales, and Nitrosomonadales in R2 were significantly higher (p b 0.05). Additionally, the abundances of Pseudomonas and (p b 0.05) than in R1 and R3. The average abundance of Rhodocyclaceae Dechloromonas (all these genera are responsible for denitrification)

Fig. 4. Heatmap analysis of the 48 dominant bacterial genera in the 12 activated sludge samples from reactor R1, R2 and R3 in different phases with increasing phenol adding in long-term operation. The sample Rx-y represented the sample collected from the x reactor in operation phase y. 8 C. Yu et al. / Science of the Total Environment 727 (2020) 138627 were higher in R2, explaining the optimal denitrifying efficiency in R2. Acknowledgements The species in the genus Dechloromonas and Pseudomonas are involved in both denitrification and polyphosphate accumulation (Jørgensen and We acknowledge the Fundamental Research Funds for the Central Pauli, 1995). Moreover, regarding phosphorous removal, the average Universities (2019QNA6013) and Science and Technology Foundation abundance of Acinetobacter, Rhodocyclaceae (such as uncultured genera of Zhejiang Province, China (2017C33020) for financing this study. Rhodocyclus), and Microlunatus in R2 were relatively higher than in R1 and R3. Concerning phenol removal, in addition to Pseudomonas and Appendix A. Supplementary data Acinetobacter, two other common phenol degraders Bacillus and Klebsi- ella were reported in R2 with SRpf acclimation. In fact, in such a system Supplementary data to this article can be found online at https://doi. where phenol and nutrients were removed, Acinetobacter, Pseudomonas org/10.1016/j.scitotenv.2020.138627. and Dechloromonas, etc., were the dominant bacterial genera regardless of whether or not SRpf was added, so we can speculate that these bacteria were involved in the removal of phenol and nutrients in the sys- References tem. Compared with the inactive SRpf control R1 or blank control R3, R2 with SRpf treatment enhanced nutrient and phenol removal. More- APHA, 2005. Standard Methods for the Examination of Water and Wastewater. American over, the relative abundances of Acinetobacter, Pseudomonas and Public Health Association, Washington, DC, USA. Blum, D., Speece, R., 1991. 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A bacterial cy- – further elucidate the mechanism of SRpf, further research is needed. tokine. Proc. Natl. Acad. Sci. U. S. A. 95, 8916 8921. Mukamolova, G.V., Murzin, A.G., Salina, E.G., Demina, G.R., Kell, D.B., Kaprelyants, A.S., Young, M., 2006. Muralytic activity of Micrococcus luteus Rpf and its relationship to CRediT authorship contribution statement physiological activity in promoting bacterial growth and resuscitation. Mol. Microbiol. 59, 84–98. Neufeld, R., Greenfield, J., Rieder, B., 1986. Temperature, cyanide and phenolic nitrification Chunna Yu:Conceptualization, Methodology, Software, Formal anal- inhibition. Water Res. 20, 633–642. ysis, Investigation, Writing - original draft, Writing - review & editing. Nikitushkin, V.D., Demina, G.R., Kaprelyants, A.S., 2011. Effect of secreted Rpf protein on Yindong Liu:Methodology, Software, Formal analysis, Investigation, intracellular contacts in Micrococcus luteus and Mycobacterium smegmatis cultures. Microbiology 80, 143–149. Writing - review & editing.Yangyang Jia:Validation, Investigation, Writ- Nikitushkin, V.D., Demina, G.R., Kaprelyants, A.S., 2016. Rpf proteins are the factors of re- ing - review & editing.Xiaomei Su:Methodology, Investigation, Writing activation of the dormant forms of Actinobacteria. Biochemistry (Mosc) 81 (13), -review&editing.Lian Lu:Formal analysis, Writing - review & editing. 1719–1734. Senoh, M., Ghosh-Banerjee, J., Ramamurthy, T., Colwell, R.R., Miyoshi, S.I., Nair, G.B., Linxian Ding:Methodology, Resources.Chaofeng Shen:Conceptualiza- Takeda, Y., 2012. Conversion of viable but nonculturable enteric bacteria to culturable tion, Methodology, Resources, Writing - review & editing, Supervision, by co-culture with eukaryotic cells. Microbiol. Immunol. 56, 342–345. Project administration, Funding acquisition. Su, X.M., Chen, X., Hu, J.X., Shen, C.F., Ding, L., 2013a. Exploring the potential environmental functions of viable but non-culturable bacteria. World J. Microb. Biot. 29, 2213–2218. Declaration of competing interest Su, X.M., Shen, H., Yao, X.Y., Ding, L.X., Yu, C.N., Shen, C.F., 2013b. A novel approach to stimulate the biphenyl-degrading potential of bacterial community from PCBs- The authors declare that they have no known competing financial contaminated soil of e-waste recycling sites. Bioresour. Technol. 146, 27–34. fl Su, X.M., Wang, Y.Y., Xue, B.B., Zhang, Y.G., Mei, R.W., Zhang, Y., et al., 2018a. Resuscitation interests or personal relationships that could have appeared to in u- of functional bacterial community for enhancing biodegradation of phenol under ence the work reported in this paper. high salinity conditions based on Rpf. Bioresour. Technol. 261, 394–402. C. Yu et al. / Science of the Total Environment 727 (2020) 138627 9

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