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

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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 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv 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, nitro- gen 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. https://doi.org/10.1016/j.scitotenv.2020.138627 0048-9697/© 2018 Elsevier B.V. All rights reserved. 2 C. Yu et al. / Science of the Total Environment 727 (2020) 138627 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.
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