New insights into filamentous sludge bulking: The potential role of extracellular polymeric substances in sludge bulking in the activated sludge process

Journal Pre-proof New insights into filamentous sludge bulking: The potential role of extracellular polymeric substances in sludge bulking in the activated sludge process Wei-Ming Li, Xi-Wen Liao, Jin-Song Guo, Yu-Xin Zhang, You-Peng Chen, Fang Fang, Peng Yan PII:

S0045-6535(20)30205-8

DOI:

https://doi.org/10.1016/j.chemosphere.2020.126012

Reference:

CHEM 126012

To appear in:

ECSN

Received Date: 15 October 2019 Revised Date:

29 December 2019

Accepted Date: 22 January 2020

Please cite this article as: Li, W.-M., Liao, X.-W., Guo, J.-S., Zhang, Y.-X., Chen, Y.-P., Fang, F., Yan, P., New insights into filamentous sludge bulking: The potential role of extracellular polymeric substances in sludge bulking in the activated sludge process, Chemosphere (2020), doi: https://doi.org/10.1016/ j.chemosphere.2020.126012. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

CRediT author statement Wei-Ming Li: Investigation, Formal analysis, Writing - Original Draft, Writing Review & Editing; Xi-Wen Liao: Investigation, Methodology, Formal analysis, Writing - Original Draft, Writing - Review & Editing; Jin-Song Guo: Conceptualization; Yu-Xin Zhang: Software; You-Peng Chen: Conceptualization; Fang Fang: Validation, Peng Yan: Writing - Original Draft, Writing - Review & Editing, Conceptualization, Resources, Project administration, Funding acquisition

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New insights into filamentous sludge bulking: the potential role of extracellular

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polymeric substances in sludge bulking in the activated sludge process

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Wei-Ming Lia, Xi-Wen Liaoa, Jin-Song Guoa, Yu-Xin Zhangb, You-Peng Chena, Fang

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Fanga, Peng Yana*

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a

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of Education, Chongqing University, Chongqing 400045, China

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b

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and Engineering, Chongqing University, Chongqing 400044, P.R. China

Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry

State Key Laboratory of Mechanical Transmissions, College of Materials Science

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*Corresponding Author

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Peng Yan

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Fax: +86-23- 65127370, Tel: +86-23- 65127370

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Email: [email protected]

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ABSTRACT

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The control of filamentous sludge bulking has been regarded as an important

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issue in the activated sludge process due to there is still a lack of understanding of the

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bulking mechanisms. In this study, changes in the extracellular polymeric substances

27

(EPS) and metabolic profile of bulking sludge based on the proteomics level was

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investigated to reveal the potential role of EPS in deteriorating sludge floc stability

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and structure during filamentous bulking. The results showed that the EPS content

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gradually decreased from 210.23 mg/g volatile suspended solids (VSS) to 131.34

31

mg/g VSS during sludge bulking. The protein (PN) content of the EPS significantly

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decreased from 173.33 mg/g VSS to 95.42 mg/g VSS during sludge bulking. However,

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a gradual increase in polysaccharides (PS) was observed. Bacterial aggregation was

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hindered by the changes in the EPS and its components. The excessive proliferation of

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filamentous bacteria had a significant effect on the molecular functions of the

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extracellular PN and metabolic pathways of the EPS. The proteins associated with the

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hydrophobic amino acid synthesis decreased, whereas the proteins associated with the

38

hydrophilic amino acid synthesis increased during sludge bulking. Electric repulsion

39

was the key factor affecting the aggregation and flocculation ability of the bacteria

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during sludge bulking. The changes in the EPS and its components induced by the

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excessive proliferation of filamentous bacteria resulted in a loose floc structure and

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poor settling performance during sludge bulking. These findings provide new insights

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into sludge bulking during the activated sludge process.

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Keywords: filamentous bulking, extracellular polymeric substances, bacterial 2

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aggregation, surface thermodynamic, proteomics

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1. Introduction

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The activated sludge process is widely used in the biological treatment of

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municipal and industrial wastewater due to its high efficiency in the removal of

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organic matter, nitrogen, and phosphorus. Filamentous bulking is a frequent problem

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in the activated sludge process and is caused by the excessive proliferation of

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filamentous bacteria (Eikelboom, 2000). The development of methods to prevent or

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control filamentous sludge bulking in wastewater treatment has been regarded as an

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important issue since the advent of the activated sludge process. Several theories were

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proposed to provide explanations for this phenomenon from the perspective of

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morphology or competition between filamentous and floccus microbes (Chudoba et

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al., 1973; Casey et al., 1994; Martins et al., 2003; Lou and de los Reyes, 2008).

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However, these theories do not entirely explain the phenomenon of filamentous

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bulking, and filamentous sludge bulking remains a serious problem in WWTPs.

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In the activated sludge process, filamentous bulking results in a loose floc

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structure of the activated sludge, an inability to form stable and dense flocs, a

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decrease in the sludge sedimentation rate, and lowered sludge compressibility

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(Jenkins et al., 1993; Li and Yang, 2007). Similarly, filamentous bacteria overgrowth

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is one of the most challenging obstacles to the structural stability of granular sludge

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and often causes the deterioration of the granular sludge properties and even

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compromises the operation (He et al., 2019). Therefore, filamentous bulking poses a

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serious threat to the stable operation of the sludge process during the long run. On the 3

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contrary, some studies have shown that granular sludge contains a large number of

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filamentous bacteria, which do not cause sludge bulking, and constitute the backbone

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of granular sludge and promote floc adhesion (Wiegant, 1988; Tay et al., 2001; Ma et

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al., 2012). Jin et al. (2003) found that flocs containing high quantities of filamentous

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bacteria settled faster than flocs without filamentous bacteria. This indicates that the

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excessive proliferation of filamentous bacteria does not necessarily lead to sludge

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bulking. Therefore, there is still controversy about the effect of filamentous bacteria

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overgrowth on microbial aggregation. The potential role of filamentous bacteria in

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microbial aggregation needs to be further revealed in both activated and granular

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sludge.

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Extracellular polymeric substances (EPS) are complex macromolecule polymers

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secreted by microorganisms, and they also originate from microbial excretion, cell

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lysis, and the adsorption of components in wastewater (Wang et al., 2013b).

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Extracellular polysaccharides (PS) and proteins (PN) are the main components of EPS.

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PS are the backbone of sludge flocs (Adav et al., 2008; Lin et al., 2013) and play an

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important role in the formation of sludge flocs (Seviour et al., 2009). PN promote

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flocs aggregation and granular sludge formation to maintain the stability of flocs

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(Flemming and Wingender, 2010; Lv et al., 2014). Therefore, PS and PN are more

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beneficial for microbial aggregation and adhesion and for increasing the stability of

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sludge flocs. Some studies found that filamentous bulking caused the changes in EPS

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contents and components in the sludge (Wang et al., 2013a; Guo et al., 2012; Guo et

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al., 2014; Moura et al., 2018; He et al., 2019). As the development of sludge bulking, 4

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much more PS and less PN were produced with a significant increase in the EPS

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amount (He et al., 2019). Guo et al. (2012) reported that the PS and PN amount

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increased during sludge bulking. There were significant differences in the changes in

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the EPS amount and its components during sludge bulking among different studies. In

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addition, the underlying mechanism of the changes in the EPS induced by filamentous

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bulking is unclear. Knowledge gaps still remain regarding the potential role of EPS in

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deteriorating the sludge floc stability and structure during filamentous bulking

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because suitable approaches for identifying the aggregation behavior of the bulking

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sludge currently do not exist. In particular, the key mechanisms determining sludge

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aggregation during filamentous bulking remain to be determined.

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The objective of the present study was to investigate the potential role of EPS in

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deteriorating the sludge floc stability and structure during filamentous bulking. The

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change in EPS, the morphological structure of the flocs, and sludge settling were

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analyzed to reveal the relationship between these factors and filamentous bacteria

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proliferation during sludge bulking. The potential of aggregation between bacteria

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was estimated quantitatively using the XDLVO theory. The metabolic profile of the

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bulking sludge was investigated at the proteomics level to elucidate the sludge

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bulking mechanism induced by EPS regulation of the filamentous bacteria. The

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results of this study provide new insights into sludge bulking during the activated

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sludge process.

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2. Materials and methods 5

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2.1. Reactor and operating conditions

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The bench-scale sequencing batch reactor (SBR) with an effective volume of 77

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L is shown in Fig. S1. The sludge of the reactor was obtained from a WWTP; the

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sewage was synthesized wastewater. The composition of the synthesized wastewater

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is presented in the Supplementary Information. The sludge retention time (SRT) of the

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SBR was 15 d and the device was operated for four cycles a day; a cycle consisted of

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10 min feeding, 110 min anoxic mixing, 180 min aerobic aeration, 50 min settling,

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and 10 min effluent withdrawal. A timer was used to automatically switch between the

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aeration, stopping, stirring, and settling states. The sludge was initially domesticated

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in the reactor under the above-mentioned operating conditions to ensure that the

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sludge had good settling performance and pollutant removal capacity. After more than

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3 SRTs, the effluent quality reached the standard and the reactor was stable for the

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subsequent experimental study. During the experiment, the dissolved oxygen (DO)

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concentration was adjusted to be as low as 0.3-0.5 mg/L to ensure that the filamentous

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sludge bulking occurred under low DO conditions. The DO was adjusted by a DO

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controller with a DO probe (Bell, BDO-200D, China) that controlled the on/off switch

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of the air compressor. With the decrease in the DO concentration, the SVI increased

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from 70 mL/g to 410 mL/g. The reactor performance during sludge bulking is shown

129

in Fig. S2.

130

2.2 Sample collection

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The samples were collected from the same SBR reactor with different SVIs (70,

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149, 230, 340, and 410 mL/g) at different times. The samples with SVIs of 70, 149, 6

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230, 340, and 410 mL/g were used for EPS extraction, floc structure analysis

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(scanning electron microscope (SEM) and DAPI-staining), contact angle, zeta

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potential analysis, XDLVO analysis, and microbial community analysis; the samples

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with SVIs of 70, 230, and 410 mL/g were used for the proteomic analysis of the

137

extracellular protein.

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2.3. Characterization of floc structure of bulking sludge

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2.3.1. Scanning electron microscope (SEM)

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The pre-treatment of the samples for SEM is described in the Supplementary

141

Information. The dried activated sludge sample was sprayed with a gold coating to

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make it conductive and improve the stability during SEM observation. The prepared

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samples were observed with a focused ion beam-SEM.

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2.3.2. DAPI-staining

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The microorganisms were stained by DAPI (4′, 6-diamidino-2-Phenylindole; 0.33

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mg/ml) for 5 min in the dark and were rinsed 3 times with a buffer solution. The glass

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slides were dried and read with an epifluorescence microscope; the details have been

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described in a previous study (Liu et al., 2001).

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2.4. Contact angle and zeta potential analysis

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The contact angle was measured according to the method described by Hou et al.

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(2015). The sludge layers were obtained by filtering the sludge suspensions onto 0.45

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µm cellulose acetate membranes with the help of a vacuum pump. Then, the layers

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were washed twice with deionized water and placed on 1% agar plate. After

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air-drying for 10 min, the sludge contact angles against the water were measured with 7

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a contact angle analyzer (DSA-100, Kruss, Germany). All contact angle values were

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based on the average value of 8 to 10 measurements.

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The zeta potentials of the sludge in different SVIs were measured by a Zeta

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meter (Zetasize Nano ZS, Malvern, UK). The sludge suspensions were obtained by

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dissolving the sludge in deionized water before the measurements. The zeta potentials

160

were measured at 25 °C. All values were based on the average value of 3 parallel

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samples.

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2.5. EPS extraction and component analysis

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The EPS were extracted using cation exchange resin (CER). About 200 mL of the

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microorganism suspension was mixed with 70 g/g volatile suspended solids (VSS)

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CER in a 500 mL conical flask and was then placed on a shaking incubator for 2 h

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with 250 rpm/min at 4 °C. The suspension was centrifuged at 10,000 g for 20 min,

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filtered through a 0.45 µm filter, and stored in the refrigerator before testing. The

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extracellular PS were determined by anthrone colorimetry (Raunkjaer et al., 1994).

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The PN content was determined by using the modified Lowry method using bovine

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serum as a standard material (Li et al., 2017).

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2.6. Microbial community and proteomic analysis

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The samples were collected and immediately stored at -20 °C. Amplicon libraries

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were used for 454 pyrosequencing and the bacterial primers 27F/533R were used for

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the V1-V3 region of the 16S rRNA gene. Proteomic analysis was used for the

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proteomic of the extracellular proteins and the treated peptides were analyzed using

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liquid chromatography-mass spectrometry (LC-MS/MS). Details on the gene 8

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pyrosequencing and shotgun proteomic assay are provided in the Supplementary

178

Information.

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2.7. Surface thermodynamics analysis and XDLVO theory

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Surface thermodynamic analysis was used to calculate the surface tension,

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interfacial tension, and surface free energy of the samples. The surface characteristics

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of the microorganisms were obtained by using the Hamaker constant ABLB and the

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interface adsorption free energy

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surface thermodynamic analysis to describe the interaction behavior between the flocs

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in the sludge-water mixtures. In the XDLVO theory, the Lewis acid-base hydration

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force (WAB) is included and the total energy of the microbial cell interaction (WT) is

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expressed as the sum of the electrostatic repulsion of the electric double layer

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interaction (WR), the van der Waals energies (WA), and (WAB): WT=WR+WA+WAB

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(Grasso et al., 2002). All of the interaction energies are related to the separation

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distance H, and are used to analyze the distance-dependent interrelationship of

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different interaction energies. The equations for the calculations are presented in the

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Supporting Information.

. DLVO theory was used in conjunction with

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3. Results and discussion

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3.1. Changes in sludge flocs during filamentous bulking

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The SEM and DAPI-staining results of the activated sludge flocs during sludge

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bulking are shown in Fig. 1. At SVI = 70 ml/g, the sludge has excellent settling

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performance. The sludge flocs are irregular with a dense structure and complex 9

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microbial population. The filamentous bacteria dispersed in the flocs are the

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framework of the sludge flocs and were attached by microorganisms such as Coccus

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and Brevibacterium. At SVI =149 ml/g, the sludge was in a limited bulking state. The

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Zoogloea covered filamentous bacteria and Coccus adhered to the filamentous

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bacteria, forming a network structure. At this stage, the filamentous bacteria were not

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dominant and they were intertwined with the Zoogloea. Only a few filamentous

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bacteria were exposed to the flocs and the sludge flocs remained stable. At SVI=230

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ml/g, the filamentous bacteria increased significantly. The filamentous bacteria

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extended out from the flocs and became longer. The adhesion of the Zoogloea to the

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filamentous bacteria decreased. The filamentous bacteria became the dominant

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bacteria and the surface of the sludge flocs was covered with a large number of

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filamentous bacteria; the sludge reached the edge of malignant filamentous bulking

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stage.

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At SVI=340 ml/g, the sludge was in the malignant bulking state. The filamentous

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bacteria grew disorderly in the sludge, and only a few bacteria adhered to it. As shown

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in Fig. 1h, the Zoogloea were loosely attached to the surface of the filamentous

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bacteria and the filamentous bacteria became longer and thinner. At SVI=410 ml/g,

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the activated sludge gradually decomposed. The sludge was almost covered by

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filamentous bacteria and only a few Zoogloeae were observed; the floc structure was

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looser, almost no other bacteria were attached, and the sludge was disintegrating.

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Based on these morphological changes, it can be inferred that the filamentous bulking

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may due to the change in the surface properties of the sludge flocs with the excessive 10

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proliferation of the filamentous bacteria; this made it difficult for the bacteria to

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aggregate and adhere to the filamentous bacteria. Finally, a dense sludge floc structure

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could not form and sludge settling was difficult, which induced the bulking of the

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filamentous bacteria in the activated sludge.

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3.2. Changes in EPS during the sludge bulking

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3.2.1. Changes in EPS components

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EPS are metabolites secreted by bacteria and relate to the population and

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metabolism of the bacteria. The excessive proliferation of filamentous bacteria

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changed the population structure of the sludge and its metabolites, thereby changing

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the EPS and its components. The EPS and its components in the sludge during sludge

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bulking were determined to assess the effects of the excessive proliferation of

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filamentous bacteria on the EPS and its components (Fig. 2a). The EPS content

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gradually decreased from 210.23 mg/g VSS to 131.34 mg/g VSS with the increase in

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the degree of sludge bulking. The EPS decreased by 37.6% during sludge bulking.

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The EPS can reduce the negative charge on the surface of the cells so that two

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adjacent cells can connect with each other (Schmidt and Ahring, 1994; Shen et al.,

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1993). When the EPS decreases, the large functional groups on the surface of the EPS

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also decrease and their ability to combine with suspended particles in wastewater

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through ion bonds, hydrogen bonds, and the van der Waals force is reduced, which

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makes it difficult to form a network structure and settling (Dignac et al., 1998).

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Extracellular polymer bridging theory states that EPS are required for the flocculation 11

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process because EPS can combine with certain parts of bacteria and particles to

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promote microbial aggregation and form activated sludge flocs. Therefore, a decrease

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in the EPS may lead to an increase in the electrostatic repulsion between the flocs, a

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lowered ability to combine with the particles, and lower settling performance, making

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it difficult for the sludge flocs to aggregate and promoting sludge bulking.

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The PN content of the EPS significantly decreased from 173.33 mg/g VSS to

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95.42 mg/g VSS with the increase in the SVI. The PN decreased by 45.0% during

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sludge bulking. As the PN decreased, the bridging effect between PN and metal ions

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decreased (Laspidou and Rittmann, 2002; Sheng et al., 2010), microorganisms had

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difficulty adhering to the sludge, and the stability of the biological aggregates

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decreased (Laspidou and Rittmann, 2002). The cross-linking network, which was

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formed as the PN attracted organic and inorganic materials, decreased and the ability

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to collect suspended particles deteriorated (Liu et al., 2004); this was not conducive to

256

sludge flocculation and settling. The amounts of hydrophobic amino acids decreased

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with the decrease in the PN, which decreased the hydrophobicity of the sludge surface,

258

increased the bound water in the sludge, and resulted in loose flocs and difficulty in

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dewatering and settling. Studies have found that hydrophobicity of the sludge surface

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is the primary factor promoting sludge flocculation (Israelachvili and Pashley, 1982;

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Pavoni et al., 1972; Struijs et al., 1991; Urbain et al., 1993; Zita and Hermansson,

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1997). This indicates that extracellular PN is the major factor in promoting sludge

263

flocculation. Therefore, a decrease in the extracellular PN content may be the key

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factor responsible for poor sludge performance and sludge bulking. 12

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With the increase in the degree of sludge bulking, the PS content of the EPS

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exhibited a gradually increasing trend. The increase in the PS may be due to the

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dominance of the filamentous bacteria during sludge bulking. Some enzyme activities

268

associated with the PS biosynthesis pathway increased and resulted in an increase in

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PS synthesis (Wang et al., 2013b). PS contain a large number of hydrophilic groups,

270

which contribute greatly to the hydrophilicity of the sludge surface (Raszka et al.,

271

2006). Therefore, the increase in PS increased the hydrophilic groups in the EPS,

272

promoted the merging of the EPS and bond water, and led to a loose sludge structure

273

and difficulty in settling.

274

3.2.2. Correlation analysis between EPS, SVI, and the abundance of filamentous

275

bacteria

276

Fig. 2b shows the correlation between the SVI, PN, PS, EPS and the abundance

277

of filamentous bacteria (FA). There was a significant correlation between the SVI and

278

EPS, indicating that the change in the EPS resulted in poor settling performance.

279

Interestingly, the abundance of filamentous bacteria (FA) was closely correlated to the

280

EPS and PN, which indicated that the excessive proliferation of filamentous bacteria

281

induced the changes in the EPS. Therefore, a hypothesis is suggested that the

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excessive proliferation of filamentous bacteria was responsible for the changes in the

283

EPS and its components, leading to poor settling performance, and thus causing

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sludge bulking. In addition, the SVI was significantly correlated with extracellular PN

285

but the correlation coefficient was lower for the PS, indicating the change in the PN

286

contributed greatly to the flocculation ability, whereas the contribution by the PS was 13

287

small. This result is consistent with the result of a previous study (Hou et al., 2015).

288 289

3.3. XDLVO analysis of sludge bulking

290

Surface thermodynamic analysis provides a quantitative description of the

291

aggregation of bacteria. The XDLVO theory was used to further explain the

292

interaction between bacteria. The results of the surface thermodynamic analysis are

293

shown in Table 1. The interfacial free energy (

294

is separated into the van der Waals interaction free energy (

295

acid-base interaction free energy (

296

adsorption between the bacteria and water molecules is stronger than the adsorption

297

between two water molecules, and hydrophilicity occurs due to the tendency of

298

bacteria adsorbing water molecules (Hou et al., 2015). As shown in Table 1,

299

were negative during the entire reaction, indicating that the

300

reactions were spontaneous and appeared as adsorption. With the increase in the

301

degree of sludge bulking, |

302

indicating that the hydrophobic interaction of the surface of the bulking sludge

303

decreased. |

304

from 63.59 mJ/m2 to 43.83 mJ/m2, indicating a decrease in the van der Waals

305

interaction and Lewis acid-base interaction.

) between the bacteria and water

) (van Oss, 1995). When

) and Lewis ＞0, the

decreased from 71.6 mJ/m2 to 45.88 mJ/m2,

| decreased from 3.01 mJ/m2 to 2.05 mJ/m2 and |

| decreased

306

The XDLVO curves of the sludge in different bulking degrees are shown in Fig. 3.

307

Most of the total energy of the interaction (WT) curves of XDLVO theory have a

308

highest point, which is called the energy barrier. The higher the barrier, the more 14

309

difficult it is for microbial particles to cross the energy barrier to cause flocculation

310

and sedimentation (Liu et al., 2010). With the increase in the degree of sludge bulking,

311

the potential energy barrier gradually increased in this study, suggesting that the

312

sludge had difficulty to aggregate.

313

In the XDLVO theory, the WT is the sum of the WA, the electric double layer

314

interaction (WR), and the acid-base interaction (WAB)：WT=WR+WA+WAB. The other

315

curves of the XDLVO theory were analyzed to obtain an insight into different

316

function forces. When the interparticle distance was between 0 nm and 1 nm, the WA

317

was predominant but actived only for a short distance (Fig. 3). With the increase in

318

the SVI, the curves of WA became sharper and the values of WA decreased, which

319

means the distance where the WA actived became shorter and the influence of the van

320

der Waals forces on the bacterial aggregation weakened in the bulking sludge. The

321

values of WAB were negative; therefore, the Lewis acid-base hydration force

322

expressed as hydrophobic interaction in the bulking sludge (Hou et al., 2015).

323

Compared with WA, the WAB actived in a longer distance, and the values of WAB

324

exhibited a significant decreasing trend. Thus, WAB was sensitive to the increase in the

325

degree of sludge bulking and the hydrophobic interaction in the bulking sludge was

326

significantly weakened. The reason may be that a decrease in the PN content in the

327

bulking sludge caused a decrease in the hydrophobic amino acid content in the PN,

328

resulting in weaker hydrophobic properties in the sludge flocs. In addition, the values

329

of WR decreased slowly and the absolute values of WR were much higher than those

330

of WA and WAB, indicating that the value of WR played an important role in the 15

331

process of sludge bulking. Thus, during sludge bulking, electric repulsion was the key

332

factor affecting the aggregation and flocculation ability of the bacteria, which led to

333

the poor setting performance.

334 335

3.4. Extracellular proteins

336

3.4.1. Functions of extracellular proteins

337

Extracellular PN are the main components of the EPS. They contain a large

338

number of enzymes and functional proteins, which play an important role in cell

339

metabolism, transport, and signal transduction. Extracellular proteomics was used to

340

further reveal the relationship between the excessive proliferation of filamentous

341

bacteria and the EPS changes based on the metabolic pathway. Using the GO database,

342

the extracellular PN were classified into three functions for annotation, namely,

343

biological processes, molecular functions, and cell components. The molecular

344

functions determine the metabolic function (Fig. 4), including catalytic activity,

345

binding activity, structural and molecular activity, transporting activity, etc. Catalytic

346

activity, binding activity, and structural molecular activity were identified as the main

347

molecular functions of the PN in the sludge with different bulking degrees. The

348

proteins involved in catalytic activity decreased from 72.4% to 67.9%, the proteins

349

involved in binding activity increased from 62.1% to 67.3%, and the proteins

350

involved in structural molecular activity increased from 3.4% to 20.5% during sludge

351

bulking. The results indicated that the excessive proliferation of filamentous bacteria

352

had a significant effect on the molecular functions of extracellular PN. Catalytic 16

353

activity is related to the catalysis of a biochemical reaction at physiological

354

temperatures. The decrease in proteins involved in the catalytic activity indicates a

355

decrease in the biochemical reaction and cell activity in the sludge. Structural

356

molecule activity is related to the structural integrity of a complex or its assembly

357

inside or outside a cell. The increase in proteins involved in structural molecule

358

activity indicates significant changes in the composition and structure inside and

359

outside of the cells.

360

The binding activity is related to the selective binding between molecules, and

361

the binding function is one of the most important molecular functions of extracellular

362

PN in the bulking sludge because the proteins with binding functions accounted for

363

62%-67% of the total. The higher the binding activity, the larger the number of

364

proteins with binding functions is and the larger the number of binding sites with

365

other molecules is (Zhang et al., 2015). The effect of the binding activity of the PN on

366

the sludge was analyzed at level 4 of GO. It is observed in Fig.5a that nucleoside

367

phosphate binding, nucleotide binding, and ribonucleotide binding are the main

368

binding functions, accounting for 20% - 40% of the binding proteins and participating

369

in almost all biochemical reactions. The proportion of nucleoside phosphate binding

370

and nucleotide binding decreased significantly from 34.5% (SVI = 70 ml/g) to 26.3%

371

(SVI = 410 ml/g) and the proportion of ribonucleotide binding decreased from 31.0%

372

to 19.6% with the increase in the degree of bulking.

373

According to the GO ID analysis (based on the UniProt database), the proteins

374

associated with nucleoside phosphate binding, nucleotide binding, and ribonucleotide 17

375

binding were very similar. The elongation factor Tu (EF-TU), 60 kDa chaperonin,

376

glycerol kinase, elongation factor G (EF-G), and ATP synthase subunit beta were

377

identified as representatives of the nucleoside phosphate, nucleotide, and

378

ribonucleotide binding proteins. Glycerol kinase and ATP synthase subunit beta are

379

proteins related to the ATP energy conversion process; ATP is involved in almost all

380

biological processes, including PN synthesis. EF-G and EF-TU are protein factors

381

involved in PN syntheses, which explains the PN reduction in the EPS. Oudshoorn et

382

al. (1999) considered EF-TU as a good indicator of the viability of bacterial cells. As

383

a co-factor for all protein translations, the decrease in EF-TU indicates a decrease in

384

cell viability. 60 kDa chaperonin promotes the correct folding and assembly of

385

proteins (according to the UniProt functional annotation) and the decrease may lead to

386

an increase in randomly folded PN, which may result in difficulty in exposure

387

hydrophobicity in EPS and thereby affect sludge settling (Jiang et al., 2010).

388

Therefore, the binding proteins affect the EPS components by reducing the PN

389

syntheses and regulating the EF-TU, ATP synthase subunit beta, etc. They also affect

390

the hydrophobicity of the EPS by reducing the exposure of the hydrophobic structure

391

by regulating the 60 kDa chaperonin. This is consistent with the findings of this study

392

related to the EPS changes; these regulating actions may be related to the metabolism

393

of enzymes.

394

Fig. 5a indicates that the cation binding proteins increased significantly.

395

Polyvalent cations have an intercellular bridging effect that promotes floc aggregation

396

(Guo et al., 2017). The increase in the cation binding proteins may be due to the 18

397

decrease in the number of cations in the EPS, which leads to the feedback-regulated

398

increase in the ability of the cells to bind to cations. The decrease in the anion binding

399

proteins was also due to the increase in the number of anions and the decrease in the

400

cell's tendency to bind to anions. In addition, the number of anion binding proteins

401

was greater than the number of cation binding proteins. Habeeb (1966) indicated that

402

anion binding proteins may have more free amino groups than cation binding proteins.

403

Therefore, the number of anion binding proteins is the key to reducing the

404

flocculation ability; this is consistent with the results of Wu et al. (2017).

405

3.4.2. Effect of protein synthesis pathway on filamentous bulking

406

In order to investigate the relationship between PN synthesis and sludge bulking,

407

the sludge samples with SVI = 230 mL/g and SVI = 410 mL/g were selected to reveal

408

the changes in the PN synthesis pathway in different bulking degrees using KEGG.

409

Nineteen pathways related to amino acid metabolism were detected as shown in Fig.

410

5b, which were related to PN synthesis (Feng et al., 2018). Alanine (Ala), valine (Val),

411

leucine (Leu), isoleucine (Ile), serine (Ser), proline (Pro), and tryptophan (Trp) are

412

hydrophobic amino acids and the others are hydrophilic amino acids. At SVI = 410

413

ml/g, the activity of the proteins of most amino acid metabolic pathways was lower

414

than at SVI=230 ml/g, which was the reason for the decrease in extracellular PN

415

(Section 3.1.1). In addition, the proteins related to some hydrophilic amino acid

416

metabolic pathways were higher, such as lysine (Lys) biosynthesis and histidine (His)

417

metabolism. The increase in the activity of the Lys biosynthesis and His metabolism

418

pathways indicates an increase in the proportion of hydrophilic amino acids in the 19

419

extracellular PN. The proteins related to Val, Leu, and Ile degradation increased more

420

than those related to biosynthesis, which indicated that the proportion of hydrophobic

421

amino acid in the extracellular PN decreased. Guo et al. (2017) believed that high

422

levels of hydrophobic amino acids in extracellular PN promoted the aggregation of

423

sludge. Therefore, the weak aggregation, poor settling performance, and the loose

424

structure of the sludge was not only the result of the decrease in extracellular PN in

425

the EPS but was also related to the decrease in hydrophobic amino acids in the

426

extracellular PN and the increase in hydrophilic amino acids.

427

3.4.3. Effect of polysaccharide synthesis pathway on filamentous bulking

428

The PS synthesis in different bulking degrees is shown in Fig. 6a. The activity of

429

the metabolic pathways associated with PS biosynthesis in the bulking sludge was

430

higher at SVI=230 mL/g and SVI=410 mL/g than at SVI=70 mL/g. Pyruvate and

431

purine are important metabolites in the cell and promote bacterial growth (Feng et al.,

432

2018) and UDP-sugar formation involved in PS synthesis (Garavito et al., 2015);

433

amino sugar and nucleotide sugar metabolism plays a key role in PS synthesis;

434

galactose (Gal) activates the secretion of PS (Kilstrup et al., 2005). The results

435

indicated an increase in the PS synthesized in the EPS and provided a reason for the

436

increase in PS, as described in Section 3.1.1. Guo et al. (2017) reported that fructose

437

and mannose metabolism are related to the synthesis of fructose 6-phosphate, which is

438

the precursor of alginate. A certain amount of alginate PS promotes the flocculation of

439

sludge (Feng et al., 2018). The activity of the fructose and mannose metabolism

440

pathway in this study was low, which may have been one of the reasons for the sludge 20

441

having difficulty to flocculate.

442

3.4.4 Correlation between the KEGG pathways and the abundance of filamentous

443

bacteria

444

In order to investigate the specific changes in the PN and PS caused by the

445

excessive proliferation of filamentous bacteria, the correlations were determined

446

between the abundance of filamentous bacteria and the pathways of synthetic PN and

447

PS. The results are presented in Fig. 6b. The highest correlations were observed

448

between the abundance of filamentous bacteria and Val, Leu, and Ile degradation, Lys

449

biosynthesis, Pyruvate metabolism, and Purine metabolism. Val, Leu, and Ile are

450

hydrophobic amino acids, whereas Lys is a hydrophilic amino acid. The results

451

indicate that the excessive proliferation of filamentous bacteria mainly affected the

452

EPS component through decreasing the hydrophobicity of extracellular PN by

453

up-regulating Val, Leu, and Ile degradation and increasing the hydrophilicity by

454

up-regulating Lys biosynthesis. Pyruvate and purine promote UDP-sugar formation

455

involved in PS synthesis. This indicates that the excessive proliferation of filamentous

456

bacteria increased PS production through promoting UDP-sugar formation by

457

up-regulating pyruvate and purine metabolism. This explains the changes in the PN

458

and PS with the increase of filamentous bacteria (Section 3.1.2). In addition, pyruvate

459

and purine participate in many critical processes, such as RNA and DNA synthesis

460

and promote filamentous bacterial growth, which explains the proliferation of

461

filamentous bacteria from a metabolic perspective.

462 21

463

3.5 The underlying mechanism of filamentous sludge bulking

464

The excessive proliferation of filamentous bacteria induced the significant

465

changes in the metabolic pathways (Fig. 6b), especially the PN and PS synthesis

466

pathway. The down-regulation of most of the PN synthesis pathways led to a decrease

467

in the PN. The up-regulation of the hydrophobic amino acid (Val, Leu, and Ile, etc.)

468

degradation pathway resulted in a decrease in the hydrophobic amino acids in the PN.

469

The up-regulation of the synthesis of some hydrophilic amino acids (Lys, His, and

470

Arg, etc.) resulted in an increase in hydrophilic amino acids in the PN. In addition, the

471

up-regulation of several (Pyruvate, Purine, and Galactose, etc.) synthesis pathway

472

related to PS synthesis resulted in an increase in PS. These changes in the PN and PS

473

led to an increase in the polar surface tension, thus Lewis acid-base interaction

474

decreased and the absolute values of WAB decreased (van Oss, 1993). The values of

475

WAB were negative and appeared as adsorption. The decrease in the absolute values of

476

WAB indicated that the adsorption of the sludge decreased during sludge bulking. In

477

addition, the decrease in PN led to a decrease in the negatively charged amino acids.

478

PN with negatively charged amino acids can bind to polyvalent cations through

479

electrostatic interaction and neutralize the negative charge on the surface of the sludge.

480

Thus, the decrease in negatively charged amino acids caused a decrease in the binding

481

capacity to the cations, and the electrostatic attractions between oppositely charged

482

poles of two particles decreased, the van der Waals interactions decreased,

483

absolute values of the WA decreased (Chen and Strevett, 2003). In addition, a decrease

484

in the neutralization of negative charges by cations binding resulted in a decrease in 22

the

485

the Zeta potential (shown in Table 1) and electrostatic repulsion increased. The WR is

486

primarily affected by the Zeta potential; the absolute values of the Zeta potential

487

increased led to the absolute values of WR decrease slowly and were much higher than

488

those of WA and WAB, indicating that the electric repulsion was the key factor

489

affecting sludge bulking. With the excessive proliferation of filamentous bacteria, the

490

total energy of the interaction (WT) gradually increased, inhibiting microbial

491

aggregation and resulting in a loose structure of the flocs, poor aggregation, and

492

deterioration of the settling performance, and ultimately causing sludge bulking.

493 494

4. Conclusions

495

The potential role of EPS in deteriorating the sludge floc stability and structure

496

during filamentous bulking was investigated in this study. The EPS and PN content

497

gradually decreased with an increase in the degree of sludge bulking. However, a

498

gradual increase in the PS content of the EPS was observed in the process. The

499

excessive proliferation of filamentous bacteria induced changes in the EPS and its

500

components and those changes were not conducive to bacterial aggregation. The

501

proteins associated with the hydrophobic amino acid synthesis decreased, whereas the

502

proteins associated with the hydrophilic amino acid synthesis increased during sludge

503

bulking. The proteins involved in the PS biosynthesis increased during sludge bulking.

504

Electric repulsion was the key factor affecting the aggregation and flocculation ability

505

of the bacteria during sludge bulking. The excessive proliferation of filamentous

506

bacteria induced changes in the EPS and its components and eventually resulted in

507

flocs with a loose structure, slow sludge settling, and poor sludge compression during 23

508

sludge bulking.

509 510

Acknowledgments

511

This work was supported by the National Natural Science Foundation of China

512

(51508546 and 51878091), the Chongqing Science and Technology Commission

513

(cstc2018jcyjAX0610), and the Fundamental Research Funds for the Central

514

Universities (2019CDQYCH036 and 2019CDXYCH0026).

515 516 517 518

Appendix A. Supplementary data E-supplementary data for this work can be found in e-version of this paper online.

519 520

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31

Table 1. Surface free energy of different degrees of sludge bulking SVI value

Contact angle (°)

Zeta potential (mv)

ABLB 10-21J

ΔG (mJ/m2)

 ΔG (mJ/m2)



ΔG (mJ/m2)

70

100.05

-16.8

2.87

-71.6

-68.59

-3.01

149

97.6

-19.1

2.63

-59.23

-56.35

-2.88

230

99.78

-21.3

2.01

-66.92

-64.8

-2.12

340

91.83

-22.1

1.92

-53.39

-51.34

-2.05

410

91.08

-22.7

2.08

-45.88

-43.83

-2.05

Figure captions Fig. 1. Scanning electron microscopy and DAPI-staining results of sludge with different degrees of bulking. Fig. 2. Changes in EPS components at different degrees of bulking (a), and correlation analysis between EPS, SVI, and the abundance of filamentous bacteria (b). Fig. 3. XDLVO curves of sludge with different degrees of bulking. Fig. 4. Classification of extracellular proteins based on molecular function. Fig. 5. Percentage of proteins associated with the binding activity in different degrees of bulking (a), and comparison of metabolic pathways related to protein synthesis in bulking sludge (b). Fig. 6. Comparison of metabolic pathway activities related to polysaccharide synthesis in bulking sludge (a), and correlation between metabolic pathways and the abundance of filamentous bacteria (b).

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Highlights • Changes in EPS were induced by excessive proliferation of filamentous bacteria. • PN significantly decreased and PS gradually increased during sludge bulking. • Increase in the surface energy led to increased difficulty in sludge aggregation. • Proliferation of filamentous bacteria affected the metabolic pathways of the EPS. • Changes in EPS resulted in a loose floc structure and poor settling.

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: