Insights into the effects of acetate on the community structure of Candidatus Accumulibacter in biological phosphorus removal system using DNA stable-isotope probing (DNA-SIP)

https://doi.org/10.1016/j.enzmictec.2020.109567Get rights and content

Highlights

  • HAc supplementation notably improved Accumulibacter and Competibacter abundance.

  • 42 -hs SIP incubation is enough for the 13C-DNA generation by Accumulibacter.

  • Clade IID exhibited the highest proportion in all clades regardless of HAc addition.

  • Accumulibacter had higher HAc assimilating activity than Competibacter.

  • Clade IIF presented the highest acetate assimilating activity in all clades.

Abstract

Sodium acetate has been most commonly used as the external carbon source to achieve successful performance of full-scale enhanced biological phosphorus removal (EBPR) processes, but its microbial mechanism for the improvement of phosphorus removal performance was still unclear. DNA based stable-isotope probing (DNA-SIP) is able to discriminate the metabolic activity of different microbes for specific substrates, thus it was applied to explore the different effects of sodium acetate on the community structure of Candidatus Accumulibacter (hereafter called Accumulibacter) and Candidatus Competibacter (hereafter called Competibacter) in a modified University of Cape Town (MUCT) process treating the real domestic sewage. Results showed that acetate addition significantly improved the abundance of Accumulibacter and Competibacter in MUCT. Accumulibacter clade IID exhibited the highest proportion in all clades before and after acetate supplementation but the proportion decreased from 95.4 % on day 23–66.3% on day 95. Contrarily, the proportion of clade IIF increased from 0.9% to 24%. DNA-SIP incubation found that the ratio of Accumulibacter in the heavy fractions to the total quantities increased faster than that of Competibacter, which successfully revealed the acetate assimilating precedence of Accumulibacter over Competibacter. Besides, the ratios of Accumulibacter clade IIF in heavy fraction increased by 22.3 %, exhibited a higher metabolic activity than other clades. Adequate acetate accomplied with high temperature possibly promoted the preferential proliferation of clade ⅡF, which provided a way to enrich clade IIF. This is the first study that successfully applied DNA-SIP to discriminate the acetate metabolic activity of Accumulibacter and Competibacter, and Accumulibacter clades.

Introduction

Enhanced biological phosphorus removal (EBPR) process, which has been widely implemented in municipal wastewater treatment plants (WWTPs), can achieve low effluent phosphorus (P) concentration by polyphosphate accumulating organisms (PAOs) undergoing alternating anaerobic/aerobic or anaerobic/anoxic mode [1,2]. PAOs absorb volatile fatty acids (VFAs) from the extracellular environment and transform them into poly-β-hydroxyalkanoates (PHAs) along with P releasing in anaerobic conditions, and achieve excessive uptake of P into cells using the energy from PHAs oxidation in anoxic or aerobic conditions. Candidatus Accumulibacter phosphatis (hereafter called Accumulibacter) is regarded as one of the dominant PAOs [3,4]. Meanwhile, glycogen accumulating organisms (GAOs) always coexist with PAOs, which can compete for short-chain VFAs with PAOs [2,5]. Candidatus Competibacter phosphatis (hereafter called Competibacter) has been widely reported as one of the main GAOs genera [6,7].

In EBPR systems, carbon sources are of paramount importance due to the direct involvement in the metabolism of PAOs and GAOs, which could notably influence the population structure of Accumulibacter and Competibacter [[8], [9], [10], [11]]. Among them, sodium acetate is one of the most widely used carbon sources in EBPR systems [10,12]. Lab-scale tests suggested that Accumulibacter could absorb acetate more rapidly than Competibacter and present a competitive advantage over Competibacter owning to its faster energy generation rate from polyphosphate (poly-P) degradation [13,14]. In WWTPs, acetate was always selected as the supplementary carbon source to improve the nutrients removal performance [10,15], however, the effects of its supplementation on the population structure of Accumulibacter and Competibacter is still unclear [16,17]. Furthermore, reports comparing the metabolic activity of Accumulibacter and Competibacter in situ are still few.

According to the diversity of polyphosphate kinase gene (ppk1), Accumulibacter is further subdivided into type I and type II, which respectively include 5 clades (IA-IE) and 9 clades (IIA-II-I) [18,19]. These clades presented diverse physiology in treatment plants, which had been extensively investigated [3,4,9,[19], [20], [21], [22]]. In order to reveal their distinct physiology, researchers were accustomed to enriching different Accumulibacter clades through synthetic wastewater [3,4,21], or focusing on the community structure and abundance under different operational conditions [19,20]. Direct comparative study on the metabolic activity of Accumulibacter clades coexisting in complex systems had been rarely reported.

DNA-based stable-isotope probing (DNA-SIP) is reported as an important culture-independent technique that can discriminate the metabolic activity of specific compounds by microbes (cultured or uncultured) in complex conditions [[23], [24], [25]]. Since its inception, DNA-SIP has been successfully employed in identifying the active ammonia-oxidizing microorganisms [26], denitrifying bacteria [27], long-chain fatty acid degrading bacteria [28] in full-scale treatment plants. In a previous study, 13C-labeled DNA-SIP was firstly used to investigate the active PAOs in an EBPR reactor treating actual wastewater [29]. However, the application of DNA-SIP in discriminating the acetate assimilating activity by Accumulibacter and Competibacter, as well as different Accumulibacter clades, has not been reported.

In this study, a typical EBPR process of modified University of Cape Town (MUCT) treating the actual domestic wastewater was operated, and acetate was selected as the external carbon source from day 33. Activated sludge collected from the MUCT system on day 110 was used to the time-course 13C-DNA-SIP incubation. Sludge samples taken from MUCT reactor and DNA-SIP incubation were analyzed using quantitative PCR (qPCR) and high-throughput sequencing, aiming to (1) analyze the community structure shifts of Accumulibacter, Competibacter and Accumulibacter clades with the acetate supplementation in the MUCT, (2) investigate the metabolic activity difference of Accumulibacter and Competibacter without the enrichment, and (3) explore the different metabolic activity of different Accumulibacter clades.

Section snippets

Experimental reactor and operation

The MUCT reactor with a volume of 72 L consisted of four zones, i.e. anaerobic zone, anoxic zone I, anoxic zone II and aerobic zone, with the volume ratio of 2:1:4:5 (Fig. 1). The seed sludge was collected from a full-scale WWTP with anaerobic-anoxic-aerobic (A2O) process. The MUCT reactor was carried out for 109 days, which was divided into two phases. In phase I (days 1–33), the actual domestic wastewater with COD concentration of 190 ± 14 mg/L was fed. In phase II (days 34–109), sodium

Performance of P removal in the MUCT system

The P and COD removal in the MUCT system over the experimental duration is shown in Fig. 2. In phase I, the MUCT was fed with the actual domestic wastewater, and presented P removal efficiency as 60.2 ± 9.7 %. The effluent P concentrations were 2.8 ± 0.6 mg/L, which were much higher than the Class 1A level (0.5 mg/L) of the Chinese discharge standard (GB18918−2002). In phase II, the supplementation of sodium acetate into the influent improved the COD concentration to 264.7 ± 47.6 mg/L, leading

Discussions

In this study, the P removal performance of MUCT became higher and more stable after acetate supplementation, but the PAM activity declined to some extent. QPCR quantification showed that the abundance of Accumulibacter and Competibacter in the MUCT system increased a lot after the acetate supplementation, and Competibacter exhibited a higher increasing extent than Accumulibacter. Nevertheless, the Competibacter abundance was still much lower than Accumulibacter (only 19 % of the Accumulibacter

Authors agreement

Dear Editor,

We declare that this manuscript entitled “Insights into the effects of acetate on the community structure of Candidatus Accumulibacter in biological phosphorus removal system using DNA stable-isotope probing (DNA-SIP)” is original, has not been published before and is not currently being considered for publication elsewhere. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but

CRediT authorship contribution statement

Baogui Wang: Conceptualization, Investigation, Data curation, Writing - original draft. Wei Zeng: Conceptualization, Resources, Supervision, Project administration, Funding acquisition, Writing - original draft. Ning Li: Investigation, Formal analysis, Software. Yu Guo: Investigation, Methodology. Qingan Meng: Investigation, Visualization. Shuo Chang: Investigation, Formal analysis. Yongzhen Peng: Supervision, Project administration, Funding acquisition.

Declarations of Competing Interest

None.

Acknowledgment

The authors would like to acknowledge the Natural Science Foundation of China (No. 51578016), the National Key Research and Development Programme of China (No. 2016YFC0401103), and the Natural Science Foundation of Beijing (No. 8172014), which provided financial support.

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