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Mg2+ distribution in activated sludge and its effects on the nitrifying activity and the characteristics of extracellular polymeric substances and sludge flocs
Journal Pre-proof Mg2+ distribution in activated sludge and its effects on the nitrifying activity and the characteristics of extracellular polymeric substances and sludge flocs Lanhe Zhang, Qiannan Zhao, Mingshuang Zhang, Jingbo Guo, Jing Zheng, Zicheng Chen, Yanping Jia, Jian Zhang, Zheng Li, Haifeng Zhang
PII:
S1359-5113(19)30700-7
DOI:
https://doi.org/10.1016/j.procbio.2019.10.002
Reference:
PRBI 11790
To appear in:
Process Biochemistry
Received Date:
14 May 2019
Revised Date:
14 August 2019
Accepted Date:
3 October 2019
Please cite this article as: Zhang L, Zhao Q, Zhang M, Guo J, Zheng J, Chen Z, Jia Y, Zhang J, Li Z, Zhang H, Mg2+ distribution in activated sludge and its effects on the nitrifying activity and the characteristics of extracellular polymeric substances and sludge flocs, Process Biochemistry (2019), doi: https://doi.org/10.1016/j.procbio.2019.10.002
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Mg2+ distribution in activated sludge and its effects on the nitrifying activity and the characteristics of extracellular polymeric substances and sludge flocs Lanhe Zhanga,b, Qiannan Zhaoa, Mingshuang Zhanga*, Jingbo Guoc, Jing Zhenga, Zicheng Chena, Yanping Jiaa, Jian Zhang a, Zheng Lia, Haifeng Zhanga* School of Chemical Engineering, Northeast Electric Power University, 132012, Jilin, China b
Key Laboratory of Songliao Aquatic Environment, Ministry of Education, Jilin Jianzhu
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a
University, 130118, Changchun, China
School of Civil and Architecture Engineering, Northeast Electric Power University,132012, Jilin, China
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Lanhe Zhang and Qiannan Zhao are co-first authors
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*Corresponding authors. E-mail addresses: [email protected] ( M. S. Zhang), [email protected]
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(H. F. Zhang)
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Graphical abstract
Highlights
Low concentration of Mg2+ (< 3 mmol/L) promotes nitrification activity of sludge.
The distribution of Mg2+ in the activated sludge floc was as follows:
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pellet>supernatant>LB-EPS>TB-EPS.
The number of groups in EPS increased with the Mg2+ concentration.
Aromatic PN-like substances and humic acid-like substances were identified in
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Abstract
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LB-EPS and TB-EPS.
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Divalent cations act as bridges among extracellular polymeric substances (EPS) and form cross-linkage for the self-immobilization of microbial biomass. However,
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their effects on the nitrification performance during the biological nitrogen removal are still unclear. In the present study, the effects of Mg2+ on the nitrifying activity, EPS and floc characteristics were investigated using a lab-scale sequencing batch reactor. The distribution of Mg2+ was quantified at different level of sludge floc. The results indicated that the nitrification activity was significantly improved when influent Mg2+ 2
was below 1.1 mmol/L, but suppressed at 3 mmol/L. The overall performance characterized by COD, NH4+-N and TN, the particle size and sludge flocculation ability rapidly increased with the increase of Mg2+ concentration. Mg2+ was mainly distributed in the pellet and changed slightly in supernatant, LB-EPS and TB-EPS. The four fluorescence peaks detected by three-dimensional excitation-emission matrix spectra were attributed to PN-like substances and humic acid-like substances in the LB-EPS
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and TB-EPS. The results of XPS analysis demonstrated that LB-EPS and TB-EPS
comprised similar elements. Therefore, the types of EPS functional groups was unchanged under varied Mg2+ concentrations, while their proportions changed and LB-
Wastewater treatment; Mg2+; Extracellular polymeric substances;
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Keywords:
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EPS/EPS was key factor for the changes of bioflocculation.
1. Introduction
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Bioflocculation; Nitrifying activity
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Activated sludge, which is composed of microbial populations, extracellular polymeric substances (EPS) secreted by bacterial metabolism and inorganic matters,
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plays a predominant role in the biological wastewater treatment processes since it can
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mineralize organic compounds due to microbial metabolism. There is a considerable interest in understanding the bacterial metabolites and the characteristics of the sludge flocs because nitrification and bioflocculation are major bottlenecks in improving the biological nitrogen removal capacity. Once the nitrifiers are washed out due to poorflocculated sludge, recovery of the nitrification capacity is time-consuming due to the slow growth rates of the nitrifiers. It is important to maintain optimal bioflocculation 3
of sludge for the nitrification process. The presence of divalent cations was reported to have positive effects on bioflocculation as they could change the metabolites, settleability and filterability of the sludge flocs [1]. EPS mainly arises from the bacterial metabolism and its major constituents include proteins, polysaccharides, humic compounds, nucleic acids and lipids [2,3]. It has great effects on the properties of microbial aggregates based on the
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adsorption, adhesion, surface charge and biodegradation characteristics. The analysis of EPS characteristics is conducive to better understand the mechanisms of bioflocculation by the divalent cations.
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Mg2+ is ubiquitous in the wastewater and it is also used as a conditioner during the
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wastewater treatment. Mg2+ enhanced the sludge microbial aggregation process through three ways: (1) Mg2+ could neutralize the negative charge on bacterial surface and
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promote the sludge aggregation [4]; (2) Mg2+ acted as a bridge and then formed EPS-
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Mg2+-EPS cross-linkage by binding to the functional groups of EPS [5]; (3) Mg2+ could interact with alkalinity and then form carbonate and phosphate precipitates [6].
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Meanwhile, Mg2+ was a necessary inorganic salt for microbial growth as it could stimulate the enzyme reactions associated with the synthesis of cell materials [7],
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especially for microorganisms with low specific growth rates such as ammonia oxidizers and nitrite oxidizers. However, it was unclear how Mg2+ affected nitrification process and enhanced the enzyme activity of nitrifiers. Therefore, the impact research of Mg2+ on nitrification activity, EPS and flocculation characteristics is of great significance for the improvement of biological wastewater treatment performance. 4
In the present study, a lab-scale sequencing batch reactor (SBR) was used to study the corresponding distribution of Mg2+ in the activated sludge. The performances of nitrogen and COD removal and the nitrification activity of sludge were explored under different Mg2+ concentration. The effect of Mg2+ on EPS was investigated by employing three-dimensional excitation-emission matrix (EEM) fluorescence spectra and X-ray photoelectron spectroscopy (XPS). In addition, the influence mechanisms of Mg2+ on
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the bioflocculation were discussed based on the above information. The results of this study are expected to provide a theoretical basis for improving the stability and
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nitrification efficiency of wastewater biological treatment systems.
2. Materials and Methods
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2.1. Experimental set-up and operation conditions
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The experiment was carried out using a lab-scale cylinder SBR with a working volume of 4.0 L (Fig. 1). The SBR was operated two cycles each day. Each cycle
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included five steps: fill (15 min), aeration (240 min), anoxic reaction (120 min), settle
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(30 min) and draw (15 min). The replacement ratio was 80% and the hydraulic retention time (HRT) was 7 h. Fine air bubbles were supplied through a diffuser at the bottom of
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the bioreactor during the aeration. Temperature was kept at 20-23°C. Dissolved oxygen (DO) and pH were monitored by Pro 20 DO meter (YSI, USA) and Five Easy PlusTM pH meter (Mettler-Toledo, China), respectively. A synthetic wastewater was used as the influent and COD, NH4+-N and PO43--P were 300 mg/L, 27 mg/L and 3 mg/L, respectively. Pure water was used as raw water
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in synthetic wastewater and the compositions of synthetic wastewater were as follows: C6H12O6·H2O (0.28 g/L), NH4Cl (0.1 g/L), KH2PO4 (0.02 g/L), NaHCO3 (0.17 g/L) and element trace solution. Trace element solution included CoCl2·6H2O (0.42 mg/L), FeCl3·6H2O (0.37 mg/L), Na2MoO4·2H2O (0.15 mg/L), CuSO4·5H2O (0.1 mg/L) and MnSO4·H2O (0.13 mg/L). The influent Mg2+ concentration was adjusted by adding magnesium sulfate (MgSO4·7H2O) and it was controlled at 0.45, 0.65, 0.9, 1.1 and 3
Fig. 1. Schematic diagram of SBR.
2.2. Analysis of conventional indicators
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mmol/L. SBR was operated for 20 days under each concentration condition.
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COD, TN, NH4+-N, SVI, MLSS and MLVSS were assayed according to the
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Standard Methods for the Examination of Water and Wastewater [8]. The particle size of sludge was measured using a particle size analyzer (Ambivalue, LFC101). The
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sludge morphology was analyzed using a fluorescence inverted biological microscope
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(Eclipse Ti-S, Nikon, Japan). The flocculability of sludge was detected using the method of Wilén et al [9]. Specially, 80 mL of sludge was transferred into a beaker
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placed on an ice bath and sonicated at an output power of 50 W for 30 s, which was sufficient to disrupt the flocs without causing cell rupture. 10 mL of suspension
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obtained by the above steps was centrifuged at 1200 rpm for 2 min and the absorbance of the supernatant was measured at 550 nm (A). The rest of the sonicated suspension was stirred for 15 min using a magnetic stirrer at a constant low speed to keep the sludge in suspension and to allow reflocculation. 10 mL of mixed liquid was centrifuged at 1200 rpm for 2 min after magnetic stirring and the absorbance of the supernatant was 6
analyzed at 550 nm (B). The flocculation ability (FA) of the sludge flocs was calculated as follows: FA
AB 100% A
Nitrification activity of activated sludge was characterized by specific oxygen uptake rate (SOUR). SOUR was determined by adding allyl thiourea (ATU) and sodium chlorate (NaClO3) to selectively inhibit the activity of ammonia-oxidizing bacteria
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(AOB) and nitrite-oxidizing bacteria (NOB). The specific measurement methods were as follows: The DO meter was inserted into the device through a perforated rubber
stopper, and the conical flask was sealed. DO was recorded one time every 30 s. DO in
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the mixed liquid of activated sludge was recorded for 3 min and its decrease value
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represented the total SOUR. NaClO3 (2.13 mg/L) was then added into the mixed liquid
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and the decrease value of DO was recorded within 3 min. At last, ATU (5 mg/L) was added into the mixed liquid and the decrease value of DO was recorded within 3 min.
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The difference between the total SOUR and the SOUR in the presence of ATU represented the SOUR of AOB (SOURAOB). The difference between the SOUR in the
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presence of NaClO3 and the SOUR in the presence of both inhibitors represented the SOUR of NOB (SOURNOB).
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Mg2+ in pellet was measured using U.S. Environment Protection Agency (1996)
Method 3050B. The sample of pellet underwent acid digestion after it was dried at 105°C. Mg2+ in the acid-digested samples was quantified using atomic absorption spectroscopy (Shimazu, Japan, AA-7000). Mg2+ in the supernatant and EPS was also measured using atomic absorption spectroscopy. 7
2.3. EPS extraction and analysis The EPS extraction protocol was modified based on the methods described by Li and Yang [10] and Yu et al. [11]. Specifically, the mixed liquid was sampled at the end of the anoxic phase and separated into four parts: supernatant, LB-EPS, TB-EPS and pellet. First, 30 mL of sludge suspension was centrifuged at 4000 g for 5 min at 4°C, and the supernatant was collected. NaCl solution (0.05% NaCl) was preheated to 70°C.
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After complete removal of the supernatant, the sludge sample was immediately
resuspended in the tube to its original volume. Meanwhile, the sludge suspension was
sheared by a vortex mixer for 60 s. Then, it was centrifuged at 4000 g for 10 min at 4°C
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and the supernatant was collected as LB-EPS. The sludge sample left in the tube was
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suspended again to 30 mL by adding NaCl solution, and then was put into a water bath at 60°C for 30 min. Furthermore, it was centrifuged at 4000 g for 15 min at 4°C. After
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centrifugation, the sample was filtered through 0.45 μm cellulose acetate membrane
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and the filtrate was collected as TB-EPS.
TOC were determined using a TOC analyzer (liqui TOC II, Elementar, Germany).
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PN was analyzed using modified Bradford method and bovine serum albumin (BSA) was used as the standard reference. PS was determined with the Anthrone-sulfuric acid
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method using glucose as the standard reference. 2.4. EEM and XPS analysis EEM spectra of EPS were measured using a luminescence spectrometer (RF-6000, Shimadzu, Japan) at excitation wavelengths of 220-400 nm and emission wavelengths of 220-550 nm by 5 nm increments. Excitation and emission slits were maintained at 5 8
nm, and the scanning speed was set at 2400 nm/min for all the measurements. The spectrum of double distilled water was recorded as the blank [12]. Part of EPS solution extracted at the end of anoxic phase was freeze-dried for XPS spectroscopy analysis. XPS spectra of all samples were acquired by using ESCALAB 250 X-ray Photoelectron Spectrometer, made in Thermo Fisher Scientific company, USA. Monochromated Al K Alpha was used as the source of X-rays (1486.71 eV) at
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72 W and 12 kV and the pass energies were 200 eV. The spot size was 500 μm and the XPS PEAK 4.1 program was employed for the data analysis. The changes of binding
energies for the elements (C, N and O) in functional group of EPS were surveyed at a
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3. Results and discussion
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pass energy of 50 eV and a stepwise energy of 0.05 eV.
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3.1. Effect of Mg2+ on the nitrification activity
Fig. 2 showed the changes of removal efficiencies of NH4+-N, TN and COD under
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different Mg2+ concentrations. The pollutants removal were positively affected by Mg2+ concentrations. The average removal efficiency of COD was stable at 90.7%. The
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average removal efficiencies of NH4+-N and TN increased from 92.3% and 65.3% to
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98.1% and 74.7%, respectively. It demonstrated that microorganisms could adapt to lower divalent ion concentrations by regulating their own metabolic systems. Low concentration of metal ions could stimulate microbial metabolic activity and improve removal capacity of pollutants [13]. The high removal efficiencies of NH4+-N and TN suggested that Mg2+ might promote the enzyme activities of nitrifiers and denitrifiers
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[14]. Fig. 2. Performances of nitrogen and COD removal.
The nitrification activity based on the SOUR method was plotted against the Mg2+ concentration (Fig 3). SOURAOB and SOURNOB increased with the increase of Mg2+ concentrations up to 1.1 mmol/L, while decreased at 3 mmol/L, which demonstrated that low concentration of Mg2+ could promote the nitrification activity. The increase of
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SOURAOB and SOURNOB could attribute to the stimulation of Mg2+ to the synthesis and
transcription of enzymes involved in the nitrification process [14]. By contrast, significant reduction of respiration rates was observed at high concentration of Mg2+ (3
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mmol/L), which was consistent with the previous studies [15,16]. There were two
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reasons for nitrification inhibition by metal ions: (1) Metals that entered cells could interact with functional groups and change the structure and function of protein [17];
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(2) Nonessential metals might displace essential metals from their metabolic sites and
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inhibit the function of various physiological cations [18,19]. Our experiments found that the removal efficiencies of NH4+-N and TN changed a little with the change of
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nitrification activity. It could be seen that microbial activity was not the main restrictive
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factor for the removal of pollutants under lower than 3 mmol/L of Mg2+ concentration. Fig. 3. Effects of Mg2+ on the nitrification activity of sludge.
It was also notable that the ratio of MLVSS/MLSS slightly increased as Mg2+
increased from 0.45 to 1.1 mmol/L, but drastically reduced at 3 mmol/L. The MLVSS/MLSS ratio profiles implied the changes of inorganic content of sludge. Under low concentration of Mg2+, its role in promoting microbial growth was greater than its 10
contribution to the inorganic component of sludge. When Mg2+ concentration was 3 mmol/L, its contribution to the inorganic component increased, which led to a severer mass transfer limitation in the activated sludge. The resistance to substrate diffusion inside the sludge increased proportionally with the increase of particle size (Fig. 9) and inorganic content, which made the substrate less available to the sludge center and eventually resulted in a substrate deficiency. Correspondingly, the sludge bioactivity
accumulation.
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3.2. The distribution of Mg2+ in the activated sludge
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became lower and the values of SOURAOB and SOURNOB decreased with Mg2+
The distribution of Mg2+ in the activated sludge was illustrated in Fig. 4. The order
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of Mg2+ concentration in the activated sludge was as follows: pellet>supernatant>LB-
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EPS>TB-EPS. Yu et al. [20] mentioned the cation distribution in excess sludge from two municipal wastewater treatment plants and found that half of Mg2+ presented in the
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supernatant and the rest of Mg2+ distributed in the EPS. Li et al. [21] investigated the distribution of cation after the flocculation of the activated sludge and demonstrated
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that bivalent cations freely distributed at the outer layer of sludge flocs. In the present
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study, Mg2+ concentration in the supernatant, LB-EPS and TB-EPS kept stable, but it appeared to be particularly high in the pellet when influent Mg2+ concentration was 3 mmol/L. Cation could accumulate in the solids as a result of salts precipitation because of its low solubility (e.g., Mg2+ interacted with alkalinity and thus formed MgCO3) [6]. Sanin et al. [22] also confirmed that potassium ions incorporated into the flocs was mainly inside the cells rather than in the EPS matrix. However, Mg2+ could be 11
incorporated into the cells as required, or it could stay in the extracellular medium of the polymer structure due to its large hydrated ion size. Fig. 4. Cations distribution in different EPS fractions.
3.3. Effect of Mg2+ on EPS composition and structure 3.3.1. Effect of Mg2+ on EPS contents
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EPS production would be substantially enhanced when the microorganisms was subject to stressful environment conditions, such as multivalent cations [23]. It was
proved that the bacteria in the activated sludge suspension and floc matrix were likely
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a dynamic double-layered EPS structure that contained loosely bound EPS (LB-EPS)
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and tightly bound EPS (TB-EPS) [24,25]. Fig. 5 a) showed the changes of EPS under different concentration of Mg2+. LB-EPS and TB-EPS were slight changed when Mg2+
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concentration increased from 0.45 to 1.1 mmol/L. LB-EPS was around 43.6 mg TOC/g VSS and TB-EPS was 45.6 mg TOC/g VSS. When Mg2+ concentration increased to 3
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mmol/L, EPS greatly reduced. LB-EPS and TB-EPS were respectively reduced to 16.6
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and 32.7 mg TOC/g VSS. It's worth noting that the stratification components of EPS changed as TB-EPS was much higher than LB-EPS. Previous studies verified that the
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ratio of LB-EPS/EPS was related to sludge flocculability and dewaterability. Particularly, LB-EPS was closely correlated with the bioflocculability and excessive LB-EPS deteriorated the sludge flocculability [26,20]. In our study, LB-EPS/EPS decreased from 49.5% to 33.6% when Mg2+ concentration increased from 0.45 to 1.1 mmol/L. FA of sludge increased and SVI declined. The decrease of LB-EPS/EPS is the
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main reason for the improvement of sludge flocculation. Fig. 5. Effects of Mg2+ on EPS, PN and PS: a) EPS, b) PN and PS of LB-EPS, c) PN and PS of TB-EPS.
PN and PS containing a large quantity of functional groups were the major components of EPS and they had a great impact on the properties of microbial aggregates [27,28]. As shown in Fig. 5 b) and c), PN and PS slightly increased when
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Mg2+ concentration increased from 0.45 to 0.65 mmol/L because it might promote the
synthesis of nucleic acids and proteins. PN declined when Mg2+ concentration increased from 0.65 to 3 mmol/L. However, Mg2+ had a slightly different effect on the content of
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PS. PS content in the TB-EPS did not significantly change, but PS content in the LB-
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EPS had a same variation tendency as PN and evidently declined under Mg2+ concentration of 3 mmol/L. Higher concentration of Mg2+ might suppress the
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production of PS, which was consistent with the finding of Sajjad and Kim [29].
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3.3.2. EEM fluorescence spectra of EPS at different Mg2+ concentration EEM fluorescence spectra of LB-EPS and TB-EPS under 0.45, 1.1 and 3 mmol/L
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of Mg2+ concentration were shown in Fig. 6. Each EEM fluorescence spectrum
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provided special information about the chemical composition of EPS. Four dominant peaks could be identified according to the method described by Chen et al. [30]. Peak A in LB-EPS and TB-EPS was located at the excitation/emission wavelengths (Ex/Em) of 255-265/315-330nm, and Peak B was situated at 220-230/325-335nm. Peak A and Peak B were assigned to tryptophan-like substances and tyrosine-like substances, respectively. Peak C in TB-EPS was identified at Ex/Em of 245-260/420-440nm, 13
corresponding to humic acid-like substances. Peak D was identified at Ex/Em of 290325/370-440nm, which was corresponded to humic acid-like substances. The tryptophan-like (Peak A) and tyrosine-like substances (Peak B) were two major substances in LB-EPS and TB-EPS. In addition, humic acid-like substances (Peak C) existed in TB-EPS was stayed at a relatively low content. The intensity of peak A and peak B increased with the increase of Mg2+ concentration, indicating that the aromatic
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protein in EPS increased. The flocculation capability of sludge improved with the
increase of aromatic protein because the aromatic protein-like substance might play an important role in maintaining the stable structure of the sludge flocs [12,31].
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Fig. 6. EEM of LB-EPS and TB-EPS under different concentration of Mg2+: a1) LB-EPS at 0.45
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mmol/L of Mg2+, b1) LB-EPS at 1.1 mmol/L of Mg2+, c1) LB-EPS at 3 mmol/L of Mg2+, a2) TBEPS at 0.45 mmol/L of Mg2+, b2) TB-EPS at 1.1 mmol/L of Mg2+, c2) TB-EPS at 3 mmol/L of Mg2+.
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Additionally, all the fluorescence peaks either shifted towards longer (red shift) or
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shorter wavelengths (blue shift) according to the emission and/or excitation scale. Compared with the fluorescence peak location of LB-EPS and TB-EPS at 0.45 mmol/L
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of Mg2+, Peak A in the TB-EPS was red-shifted by 10 nm along the Ex axis under 1.1 and 3 mmol/L of Mg2+, which might be attributed to the increase of carbonyl-containing
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substituents, hydroxyl, amino groups and carboxyl constituents [32]. Peak B in the LBEPS was blue-shifted by 5 nm along the Em axis under 3 mmol/L of Mg2+, and that in the TB-EPS was blue-shifted 10 nm along the Ex axis and red-shifted by 5 nm along the Em axis under 1.1 mmol/L of Mg2+. The shift of Peak B could be associated with the reduction of some functional groups, such as aromatic ring and conjugated bond in 14
chain structures [33]. All the shifts of the fluorescence peaks could provide information on the changes of chemical structure, which would indicate the functional group change of EPS under different Mg2+ concentration. A significant increase of FA was observed under 1.1 and 3 mmol/L of Mg2+ concentration, which indicated that an increase of hydroxyl, amino and carboxyl constituents favored the bioflocculation.
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3.3.3. XPS analysis of EPS at different Mg2+ concentration Fig. 7 a) showed the X-ray photoelectron spectra of EPS obtained in the energy range of 0-1200 eV (1.0 eV step-size). Both LB-EPS and TB-EPS comprised similar
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elements under different Mg2+ concentration. The main elements were C, N and O that participated in the adsorption of Mg2+.
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The changes of C 1s and O 1s functionalities in the LB-EPS and TB-EPS under
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different Mg2+ concentration were investigated to obtain more detailed information on the chemical bonding states of the elements (0.05 eV step size). The resulting peaks
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were later resolved into their individual components. High resolution spectra were shown in Fig. 7 b-e) only under 0.45 mmol/L of Mg2+ since XPS spectra were large
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homologous in terms of functionalities of EPS. Consequently, peak decomposition
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looked very similar and the chemical functional groups (molar ratio with respect to total carbon) were listed in Table 1. Fig. 7. a) XPS wide survey scans of LB-EPS and TB-EPS under different concentration of Mg2+
and XPS narrowly scanned spectra of b) C 1s of LB-EPS, c) C 1s of TB-EPS, d) O 1s of LB-EPS and e) O 1s of LB-EPS under 0.45 mmol/L of Mg2+.
The C 1s peak in both LB-EPS and TB-EPS was resolved into four component 15
peaks: (1) 284.8 eV C-(C,H) from hydrocarbons; (2) 286.2 eV C-(O,N) from proteins and alcohols; (3) 287.8 eV C=O or O-C-O from carboxylate, carbonyl, amide, acetals or hemiacetals; (4) 288.7 eV O=C-OR from uronic acids. The O 1s peak was decomposed into two peaks at 531.3 eV (O=C from carboxylate, carbonyl, ester, or amide) and 532.7 eV (O-(C,H) from hydroxide, acetals or hemiacetals). Only nonprotonated nitrogen compounds were detected in EPS, which indicated that amides
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(peptides) were dominant [34]. The peak pertaining to C=O was more prevalent in TBEPS (as shown in Fig. 8 and Table 1). However, the second O 1s component located at 531.3 eV presented an opposite situation. The binding energy of the two kinds of O was
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unchanged with the variation of Mg2+ concentration, which indicated that their
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chemical environment was similar and they did not participate in the chemical adsorption of Mg2+. An apparent trend could be observed in Table 2 that carboxylate
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(O531.3) contents of EPS increased with the addition of Mg2+ due to decreasing
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contribution from hemiacetals, acetals and alcohols (O532.7). C=O groups were often found in protein secondary structures and certain protein secondary structures promoted
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bioflocculation [35]. In our study, the proportions of carboxylate (O531.3) increased in EPS, which might the reasons for the enhancement of bioflocculation.
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Table 1. Functional group compositions obtained from narrowly scanned spectra of XPS.
3.4. Floc characteristics analysis of the activated sludge SVI and FA could characterize the settleability and flocculation of the sludge. The sludge growth was explored by measuring the sludge particle size. Fig. 8 a) showed that the average particle size of the sludge increased gradually with the increase of Mg2+ 16
concentration. When Mg2+ concentration increased from 0.45 to 3 mmol/L, FA increased from 15.4% to 29.2% and SVI decreased from 52.8 to 35.2 ml/g. The flocculation and settleability of activated sludge flocs were obviously improved with the increase of Mg2+ concentration. At the same time, the increase of Mg2+ concentration resulted in the change of sludge morphology from loose structured flocculent sludge to large and dense sludge particles (Fig. 8 b), c)). The ratios of
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extracellular PN/PS were related to the characteristics of bioflocs and biogranules
[36,37]. Aerobic bioflocs with a higher PN/PS ratio had a poor settleability [38,39]. In our work, PN/PS dropped from 3.5 to 1.2 when Mg2+ concentration increased from 0.45
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to 3 mmol/L (Section 2.3.1). SVI was in the range of 34.1-52.8 ml/g, which seemed to
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be lower than that reported by Tay et al. [37]. It was because there was another potential factor affecting the settleability because Mg2+ increased the inorganic composition of
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floc. With the continuous increase of Mg2+ concentration, sludge flocs became denser
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and more compact [40]. Sludge flocs with poor settleability was washed out in the initial period due to short settling time, and then resulted in a low SVI value.
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Fig. 8. Effects of Mg2+ on the properties and morphology of sludge: a) sludge properties, b) sludge
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morphology at 0.45 mmol/L of Mg2+ (objective×40), c) sludge morphology at 3 mmol/L of Mg2+ (objective×40).
According to 3.2 and 3.3, the increase of Mg2+ concentration in the pellet was more
obvious than that in EPS. Both EEM and XPS results showed that the changes of Mg2+ concentration did not change the components and functional groups of EPS, but changed their proportions. Thus, Mg2+ concentration affected the flocculation and 17
settleablilty of sludge by changing the content of protein and polysaccharide in the EPS. Furthermore, the addition of Mg2+ increased the density of the flocs and the maximum SVI declined. The constant content of Mg2+ in the supernatant indicated that the neutralization of negative charge was not the main reason for the increase of bioflocculation.
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4. Conclusions The effects of Mg2+ concentration on the nitrifying activity, EPS and floc
characteristics of the activated sludge were investigated and the conclusions were as
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follows:
(1) The nitrification activity of the activated sludge promoted under low
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concentrations of Mg2+, while reduced under higher concentrations of Mg2+. When
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Mg2+ concentration was 1.1 mmol/L, the reactor had the optimal pollutant removal capacity and nitrification activity. Meanwhile, the sludge had good bioflocculation and
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sedimentation.
(2) The order of Mg2+ concentration in the activated sludge was as follows:
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pellet>supernatant>LB-EPS>TB-EPS. Mg2+ concentration in supernatant, LB-EPS and
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TB-EPS changed slightly, whereas significant changes were observed in the pellet. (3) High concentration of Mg2+ (3 mmol/L) reduced the EPS concentration and
changed its stratification components, while improved the bioflocculation and sedimentation performance of sludge and increased sludge particle size. The decrease of LB-EPS was the main reason for the improvement of bioflocculation. The increase of inorganic components and sludge particle size contributed to the enhancement of 18
settleability of the activated sludge. (4) The increase of Mg2+ concentration had slight influences on the type of fluorophores, but changed obviously their contents. The most components of LB-EPS and TB-EPS were similar and aromatic PN-like substances and humic acid-like substances were identified. The position of the fluorescent peak shifts and the chemical structure of EPS changed accordingly.
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Acknowledgements
The authors thank to the National Natural Science Foundation of China (grant
of
Jilin
Province
(grant
numbers
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numbers 51678119 and 51808254), the Science and Technology Development Program 20180201016SF,
20180101079JC
and
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20180101309JC) and the Science and Technology Research Project from Jilin
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Provincial Department of Education (grant numbers JJKH20180453KJ and JJKH20180454KJ) for their financial supports.
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effect on thermodynamic properties of sludge, Biochemical Engineering Journal 147 (2019) 146-152. [26] S.F. Yang, X.Y. Li, Influences of extracellular polymeric substances (EPS) on the characteristics of activated sludge under non-steady-state conditions, Process Biochemistry 44 (2009) 91-96. [27] Z.P. Wang, L.L. Liu, J. Yao, W.M. Cai, Effects of extracellular polymeric
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microorganisms under glucose-controlled conditions, Water Research 44 (2010)
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[37] J.H. Tay, Q.S. Liu, Y. Liu, The role of cellular polysaccharides in the formation and stability of aerobic granules, Letters in Applied Microbiology 33 (2010) 222-226.
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[38] D.J. Batstone, J. Keller, Variation of bulk properties of anaerobic granules with wastewater type, Water Research 35 (2001) 1723-1729.
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[39] F. Mart í nez, J. Lema, R. M é ndez, F. Cuervo-L ó pez, J. G ó mez, Role of exopolymeric protein on the settleability of nitrifying sludges, Bioresource Technology 94 (2004) 43-48. [40] X.M. Li, Q.Q. Liu, Q. Yang, L. Guo, G.M. Zeng, J.M. Hu, W. Zheng, Enhanced aerobic sludge granulation in sequencing batch reactor by Mg2+ augmentation, 24
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Bioresource Technology 99 (2009) 64-67.
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Fig. 1. Schematic diagram of SBR. Fig. 2. Performances of nitrogen and COD removal. Fig. 3. Effects of Mg2+ on the nitrification activity of sludge. Fig. 4. Cations distribution in different EPS fractions. Fig. 5. Effects of Mg2+ on EPS, PN and PS: a) EPS, b) PN and PS of LB-EPS, c) PN and PS of TB-EPS.
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Fig. 6. EEM of LB-EPS and TB-EPS under different concentration of Mg2+: a1) LBEPS at 0.45 mmol/L of Mg2+, b1) LB-EPS at 1.1 mmol/L of Mg2+, c1) LB-EPS at 3
mmol/L of Mg2+, a2) TB-EPS at 0.45 mmol/L of Mg2+, b2) TB-EPS at 1.1 mmol/L of
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Mg2+, c2) TB-EPS at 3 mmol/L of Mg2+.
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Fig. 7. a) XPS wide survey scans of LB-EPS and TB-EPS under different concentration of Mg2+ and XPS narrowly scanned spectra of b) C 1s of LB-EPS, c) C 1s of TB-EPS,
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d) O 1s of LB-EPS and e) O 1s of LB-EPS under 0.45 mmol/L of Mg2+.
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Table 1. Functional group compositions obtained from narrowly scanned spectra of XPS.
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Fig. 8. Effects of Mg2+ on the properties and morphology of sludge: a) sludge properties, b) sludge morphology at 0.45 mmol/L of Mg2+ (objective×40), c) sludge morphology
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at 3 mmol/L of Mg2+ (objective×40).
26
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Figures and Tables
1. Electromagnetic stirrer 2. Rotameter 3. Air pump 4. Influent pump 5. Air diffuser 6. Drain valve 7. Sample port 8. Dissolved oxygen meter 9. pH meter
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Fig.1. Schematic diagram of SBR.
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Fig. 2. Performances of nitrogen and COD removal.
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Fig. 3. Effects of Mg2+ on the nitrification activity of sludge.
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Fig. 4. Mg2+ distribution in pellet, TB-EPS, LB-EPS and supernatant.
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Fig. 5. Effects of Mg2+ on EPS, PN and PS: a) EPS, b) PN and PS of LB-EPS, c) PN and PS of TB-
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EPS.
31
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Fig. 6. EEM of LB-EPS and TB-EPS under different concentration of Mg2+: a1) LB-EPS at 0.45 mmol/L of Mg2+, b1) LB-EPS at 1.1 mmol/L of Mg2+, c1) LB-EPS at 3 mmol/L of Mg2+, a2) TB-
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EPS at 0.45 mmol/L of Mg2+, b2) TB-EPS at 1.1 mmol/L of Mg2+, c2) TB-EPS at 3 mmol/L of Mg2+.
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Fig. 7. a) XPS wide survey scans of LB-EPS and TB-EPS under different concentration of Mg2+
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and XPS narrowly scanned spectra of b) C 1s of LB-EPS, c) C 1s of TB-EPS, d) O 1s of LB-EPS
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and e) O 1s of LB-EPS under 0.45 mmol/L of Mg2+.
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Table 1. Functional group compositions obtained from narrowly scanned spectra of XPS. Element
Atomic (%)
Peak (eV)
Atomic (%)
Assignments
a1
a2
a3
b1
b2
b3
284.8
82.70
61.08
66.21
58.23
65.50
58.05
C-(C,H)
286.2
7.77
31.95
23.47
22.79
22.96
20.62
C-(O,N)
287.8
5.00
0.40
3.83
17.7
2.88
15.41
C=O+O-C-O
288.7
4.46
6.57
6.49
1.27
8.66
2.48
O=C-OH
531.3
11.50
34.75
48.27
28.22
41.78
69.78
O=C
532.7
88.49
65.24
51.73
71.78
58.82
30.32
C-OH; C-O-C
C 1s
O 1s
Notes: a1, a2 and a3 corresponding to LB-EPS at Mg2+ concentration of 0.45, 1.1 and 3 mmol/L,
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respectively. b1, b2 and b3 corresponding to TB-EPS at Mg2+ concentration of 0.45, 1.1 and 3
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mmol/L, respectively.
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Fig. 8. Effects of Mg2+ on the properties and morphology of sludge: a) sludge properties, b) sludge
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(objective×40).
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morphology at 0.45 mmol/L of Mg2+ (objective×40), c) sludge morphology at 3 mmol/L of Mg2+
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PII:
S1359-5113(19)30700-7
DOI:
https://doi.org/10.1016/j.procbio.2019.10.002
Reference:
PRBI 11790
To appear in:
Process Biochemistry
Received Date:
14 May 2019
Revised Date:
14 August 2019
Accepted Date:
3 October 2019
Please cite this article as: Zhang L, Zhao Q, Zhang M, Guo J, Zheng J, Chen Z, Jia Y, Zhang J, Li Z, Zhang H, Mg2+ distribution in activated sludge and its effects on the nitrifying activity and the characteristics of extracellular polymeric substances and sludge flocs, Process Biochemistry (2019), doi: https://doi.org/10.1016/j.procbio.2019.10.002
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. © 2019 Published by Elsevier.
Mg2+ distribution in activated sludge and its effects on the nitrifying activity and the characteristics of extracellular polymeric substances and sludge flocs Lanhe Zhanga,b, Qiannan Zhaoa, Mingshuang Zhanga*, Jingbo Guoc, Jing Zhenga, Zicheng Chena, Yanping Jiaa, Jian Zhang a, Zheng Lia, Haifeng Zhanga* School of Chemical Engineering, Northeast Electric Power University, 132012, Jilin, China b
Key Laboratory of Songliao Aquatic Environment, Ministry of Education, Jilin Jianzhu
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a
University, 130118, Changchun, China
School of Civil and Architecture Engineering, Northeast Electric Power University,132012, Jilin, China
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c
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Lanhe Zhang and Qiannan Zhao are co-first authors
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*Corresponding authors. E-mail addresses: [email protected] ( M. S. Zhang), [email protected]
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(H. F. Zhang)
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Graphical abstract
Highlights
Low concentration of Mg2+ (< 3 mmol/L) promotes nitrification activity of sludge.
The distribution of Mg2+ in the activated sludge floc was as follows:
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pellet>supernatant>LB-EPS>TB-EPS.
The number of groups in EPS increased with the Mg2+ concentration.
Aromatic PN-like substances and humic acid-like substances were identified in
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Abstract
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LB-EPS and TB-EPS.
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Divalent cations act as bridges among extracellular polymeric substances (EPS) and form cross-linkage for the self-immobilization of microbial biomass. However,
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their effects on the nitrification performance during the biological nitrogen removal are still unclear. In the present study, the effects of Mg2+ on the nitrifying activity, EPS and floc characteristics were investigated using a lab-scale sequencing batch reactor. The distribution of Mg2+ was quantified at different level of sludge floc. The results indicated that the nitrification activity was significantly improved when influent Mg2+ 2
was below 1.1 mmol/L, but suppressed at 3 mmol/L. The overall performance characterized by COD, NH4+-N and TN, the particle size and sludge flocculation ability rapidly increased with the increase of Mg2+ concentration. Mg2+ was mainly distributed in the pellet and changed slightly in supernatant, LB-EPS and TB-EPS. The four fluorescence peaks detected by three-dimensional excitation-emission matrix spectra were attributed to PN-like substances and humic acid-like substances in the LB-EPS
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and TB-EPS. The results of XPS analysis demonstrated that LB-EPS and TB-EPS
comprised similar elements. Therefore, the types of EPS functional groups was unchanged under varied Mg2+ concentrations, while their proportions changed and LB-
Wastewater treatment; Mg2+; Extracellular polymeric substances;
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Keywords:
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EPS/EPS was key factor for the changes of bioflocculation.
1. Introduction
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Bioflocculation; Nitrifying activity
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Activated sludge, which is composed of microbial populations, extracellular polymeric substances (EPS) secreted by bacterial metabolism and inorganic matters,
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plays a predominant role in the biological wastewater treatment processes since it can
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mineralize organic compounds due to microbial metabolism. There is a considerable interest in understanding the bacterial metabolites and the characteristics of the sludge flocs because nitrification and bioflocculation are major bottlenecks in improving the biological nitrogen removal capacity. Once the nitrifiers are washed out due to poorflocculated sludge, recovery of the nitrification capacity is time-consuming due to the slow growth rates of the nitrifiers. It is important to maintain optimal bioflocculation 3
of sludge for the nitrification process. The presence of divalent cations was reported to have positive effects on bioflocculation as they could change the metabolites, settleability and filterability of the sludge flocs [1]. EPS mainly arises from the bacterial metabolism and its major constituents include proteins, polysaccharides, humic compounds, nucleic acids and lipids [2,3]. It has great effects on the properties of microbial aggregates based on the
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adsorption, adhesion, surface charge and biodegradation characteristics. The analysis of EPS characteristics is conducive to better understand the mechanisms of bioflocculation by the divalent cations.
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Mg2+ is ubiquitous in the wastewater and it is also used as a conditioner during the
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wastewater treatment. Mg2+ enhanced the sludge microbial aggregation process through three ways: (1) Mg2+ could neutralize the negative charge on bacterial surface and
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promote the sludge aggregation [4]; (2) Mg2+ acted as a bridge and then formed EPS-
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Mg2+-EPS cross-linkage by binding to the functional groups of EPS [5]; (3) Mg2+ could interact with alkalinity and then form carbonate and phosphate precipitates [6].
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Meanwhile, Mg2+ was a necessary inorganic salt for microbial growth as it could stimulate the enzyme reactions associated with the synthesis of cell materials [7],
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especially for microorganisms with low specific growth rates such as ammonia oxidizers and nitrite oxidizers. However, it was unclear how Mg2+ affected nitrification process and enhanced the enzyme activity of nitrifiers. Therefore, the impact research of Mg2+ on nitrification activity, EPS and flocculation characteristics is of great significance for the improvement of biological wastewater treatment performance. 4
In the present study, a lab-scale sequencing batch reactor (SBR) was used to study the corresponding distribution of Mg2+ in the activated sludge. The performances of nitrogen and COD removal and the nitrification activity of sludge were explored under different Mg2+ concentration. The effect of Mg2+ on EPS was investigated by employing three-dimensional excitation-emission matrix (EEM) fluorescence spectra and X-ray photoelectron spectroscopy (XPS). In addition, the influence mechanisms of Mg2+ on
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the bioflocculation were discussed based on the above information. The results of this study are expected to provide a theoretical basis for improving the stability and
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nitrification efficiency of wastewater biological treatment systems.
2. Materials and Methods
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2.1. Experimental set-up and operation conditions
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The experiment was carried out using a lab-scale cylinder SBR with a working volume of 4.0 L (Fig. 1). The SBR was operated two cycles each day. Each cycle
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included five steps: fill (15 min), aeration (240 min), anoxic reaction (120 min), settle
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(30 min) and draw (15 min). The replacement ratio was 80% and the hydraulic retention time (HRT) was 7 h. Fine air bubbles were supplied through a diffuser at the bottom of
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the bioreactor during the aeration. Temperature was kept at 20-23°C. Dissolved oxygen (DO) and pH were monitored by Pro 20 DO meter (YSI, USA) and Five Easy PlusTM pH meter (Mettler-Toledo, China), respectively. A synthetic wastewater was used as the influent and COD, NH4+-N and PO43--P were 300 mg/L, 27 mg/L and 3 mg/L, respectively. Pure water was used as raw water
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in synthetic wastewater and the compositions of synthetic wastewater were as follows: C6H12O6·H2O (0.28 g/L), NH4Cl (0.1 g/L), KH2PO4 (0.02 g/L), NaHCO3 (0.17 g/L) and element trace solution. Trace element solution included CoCl2·6H2O (0.42 mg/L), FeCl3·6H2O (0.37 mg/L), Na2MoO4·2H2O (0.15 mg/L), CuSO4·5H2O (0.1 mg/L) and MnSO4·H2O (0.13 mg/L). The influent Mg2+ concentration was adjusted by adding magnesium sulfate (MgSO4·7H2O) and it was controlled at 0.45, 0.65, 0.9, 1.1 and 3
Fig. 1. Schematic diagram of SBR.
2.2. Analysis of conventional indicators
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mmol/L. SBR was operated for 20 days under each concentration condition.
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COD, TN, NH4+-N, SVI, MLSS and MLVSS were assayed according to the
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Standard Methods for the Examination of Water and Wastewater [8]. The particle size of sludge was measured using a particle size analyzer (Ambivalue, LFC101). The
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sludge morphology was analyzed using a fluorescence inverted biological microscope
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(Eclipse Ti-S, Nikon, Japan). The flocculability of sludge was detected using the method of Wilén et al [9]. Specially, 80 mL of sludge was transferred into a beaker
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placed on an ice bath and sonicated at an output power of 50 W for 30 s, which was sufficient to disrupt the flocs without causing cell rupture. 10 mL of suspension
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obtained by the above steps was centrifuged at 1200 rpm for 2 min and the absorbance of the supernatant was measured at 550 nm (A). The rest of the sonicated suspension was stirred for 15 min using a magnetic stirrer at a constant low speed to keep the sludge in suspension and to allow reflocculation. 10 mL of mixed liquid was centrifuged at 1200 rpm for 2 min after magnetic stirring and the absorbance of the supernatant was 6
analyzed at 550 nm (B). The flocculation ability (FA) of the sludge flocs was calculated as follows: FA
AB 100% A
Nitrification activity of activated sludge was characterized by specific oxygen uptake rate (SOUR). SOUR was determined by adding allyl thiourea (ATU) and sodium chlorate (NaClO3) to selectively inhibit the activity of ammonia-oxidizing bacteria
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(AOB) and nitrite-oxidizing bacteria (NOB). The specific measurement methods were as follows: The DO meter was inserted into the device through a perforated rubber
stopper, and the conical flask was sealed. DO was recorded one time every 30 s. DO in
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the mixed liquid of activated sludge was recorded for 3 min and its decrease value
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represented the total SOUR. NaClO3 (2.13 mg/L) was then added into the mixed liquid
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and the decrease value of DO was recorded within 3 min. At last, ATU (5 mg/L) was added into the mixed liquid and the decrease value of DO was recorded within 3 min.
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The difference between the total SOUR and the SOUR in the presence of ATU represented the SOUR of AOB (SOURAOB). The difference between the SOUR in the
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presence of NaClO3 and the SOUR in the presence of both inhibitors represented the SOUR of NOB (SOURNOB).
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Mg2+ in pellet was measured using U.S. Environment Protection Agency (1996)
Method 3050B. The sample of pellet underwent acid digestion after it was dried at 105°C. Mg2+ in the acid-digested samples was quantified using atomic absorption spectroscopy (Shimazu, Japan, AA-7000). Mg2+ in the supernatant and EPS was also measured using atomic absorption spectroscopy. 7
2.3. EPS extraction and analysis The EPS extraction protocol was modified based on the methods described by Li and Yang [10] and Yu et al. [11]. Specifically, the mixed liquid was sampled at the end of the anoxic phase and separated into four parts: supernatant, LB-EPS, TB-EPS and pellet. First, 30 mL of sludge suspension was centrifuged at 4000 g for 5 min at 4°C, and the supernatant was collected. NaCl solution (0.05% NaCl) was preheated to 70°C.
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After complete removal of the supernatant, the sludge sample was immediately
resuspended in the tube to its original volume. Meanwhile, the sludge suspension was
sheared by a vortex mixer for 60 s. Then, it was centrifuged at 4000 g for 10 min at 4°C
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and the supernatant was collected as LB-EPS. The sludge sample left in the tube was
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suspended again to 30 mL by adding NaCl solution, and then was put into a water bath at 60°C for 30 min. Furthermore, it was centrifuged at 4000 g for 15 min at 4°C. After
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centrifugation, the sample was filtered through 0.45 μm cellulose acetate membrane
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and the filtrate was collected as TB-EPS.
TOC were determined using a TOC analyzer (liqui TOC II, Elementar, Germany).
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PN was analyzed using modified Bradford method and bovine serum albumin (BSA) was used as the standard reference. PS was determined with the Anthrone-sulfuric acid
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method using glucose as the standard reference. 2.4. EEM and XPS analysis EEM spectra of EPS were measured using a luminescence spectrometer (RF-6000, Shimadzu, Japan) at excitation wavelengths of 220-400 nm and emission wavelengths of 220-550 nm by 5 nm increments. Excitation and emission slits were maintained at 5 8
nm, and the scanning speed was set at 2400 nm/min for all the measurements. The spectrum of double distilled water was recorded as the blank [12]. Part of EPS solution extracted at the end of anoxic phase was freeze-dried for XPS spectroscopy analysis. XPS spectra of all samples were acquired by using ESCALAB 250 X-ray Photoelectron Spectrometer, made in Thermo Fisher Scientific company, USA. Monochromated Al K Alpha was used as the source of X-rays (1486.71 eV) at
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72 W and 12 kV and the pass energies were 200 eV. The spot size was 500 μm and the XPS PEAK 4.1 program was employed for the data analysis. The changes of binding
energies for the elements (C, N and O) in functional group of EPS were surveyed at a
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3. Results and discussion
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pass energy of 50 eV and a stepwise energy of 0.05 eV.
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3.1. Effect of Mg2+ on the nitrification activity
Fig. 2 showed the changes of removal efficiencies of NH4+-N, TN and COD under
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different Mg2+ concentrations. The pollutants removal were positively affected by Mg2+ concentrations. The average removal efficiency of COD was stable at 90.7%. The
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average removal efficiencies of NH4+-N and TN increased from 92.3% and 65.3% to
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98.1% and 74.7%, respectively. It demonstrated that microorganisms could adapt to lower divalent ion concentrations by regulating their own metabolic systems. Low concentration of metal ions could stimulate microbial metabolic activity and improve removal capacity of pollutants [13]. The high removal efficiencies of NH4+-N and TN suggested that Mg2+ might promote the enzyme activities of nitrifiers and denitrifiers
9
[14]. Fig. 2. Performances of nitrogen and COD removal.
The nitrification activity based on the SOUR method was plotted against the Mg2+ concentration (Fig 3). SOURAOB and SOURNOB increased with the increase of Mg2+ concentrations up to 1.1 mmol/L, while decreased at 3 mmol/L, which demonstrated that low concentration of Mg2+ could promote the nitrification activity. The increase of
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SOURAOB and SOURNOB could attribute to the stimulation of Mg2+ to the synthesis and
transcription of enzymes involved in the nitrification process [14]. By contrast, significant reduction of respiration rates was observed at high concentration of Mg2+ (3
-p
mmol/L), which was consistent with the previous studies [15,16]. There were two
re
reasons for nitrification inhibition by metal ions: (1) Metals that entered cells could interact with functional groups and change the structure and function of protein [17];
lP
(2) Nonessential metals might displace essential metals from their metabolic sites and
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inhibit the function of various physiological cations [18,19]. Our experiments found that the removal efficiencies of NH4+-N and TN changed a little with the change of
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nitrification activity. It could be seen that microbial activity was not the main restrictive
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factor for the removal of pollutants under lower than 3 mmol/L of Mg2+ concentration. Fig. 3. Effects of Mg2+ on the nitrification activity of sludge.
It was also notable that the ratio of MLVSS/MLSS slightly increased as Mg2+
increased from 0.45 to 1.1 mmol/L, but drastically reduced at 3 mmol/L. The MLVSS/MLSS ratio profiles implied the changes of inorganic content of sludge. Under low concentration of Mg2+, its role in promoting microbial growth was greater than its 10
contribution to the inorganic component of sludge. When Mg2+ concentration was 3 mmol/L, its contribution to the inorganic component increased, which led to a severer mass transfer limitation in the activated sludge. The resistance to substrate diffusion inside the sludge increased proportionally with the increase of particle size (Fig. 9) and inorganic content, which made the substrate less available to the sludge center and eventually resulted in a substrate deficiency. Correspondingly, the sludge bioactivity
accumulation.
-p
3.2. The distribution of Mg2+ in the activated sludge
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became lower and the values of SOURAOB and SOURNOB decreased with Mg2+
The distribution of Mg2+ in the activated sludge was illustrated in Fig. 4. The order
re
of Mg2+ concentration in the activated sludge was as follows: pellet>supernatant>LB-
lP
EPS>TB-EPS. Yu et al. [20] mentioned the cation distribution in excess sludge from two municipal wastewater treatment plants and found that half of Mg2+ presented in the
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supernatant and the rest of Mg2+ distributed in the EPS. Li et al. [21] investigated the distribution of cation after the flocculation of the activated sludge and demonstrated
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that bivalent cations freely distributed at the outer layer of sludge flocs. In the present
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study, Mg2+ concentration in the supernatant, LB-EPS and TB-EPS kept stable, but it appeared to be particularly high in the pellet when influent Mg2+ concentration was 3 mmol/L. Cation could accumulate in the solids as a result of salts precipitation because of its low solubility (e.g., Mg2+ interacted with alkalinity and thus formed MgCO3) [6]. Sanin et al. [22] also confirmed that potassium ions incorporated into the flocs was mainly inside the cells rather than in the EPS matrix. However, Mg2+ could be 11
incorporated into the cells as required, or it could stay in the extracellular medium of the polymer structure due to its large hydrated ion size. Fig. 4. Cations distribution in different EPS fractions.
3.3. Effect of Mg2+ on EPS composition and structure 3.3.1. Effect of Mg2+ on EPS contents
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EPS production would be substantially enhanced when the microorganisms was subject to stressful environment conditions, such as multivalent cations [23]. It was
proved that the bacteria in the activated sludge suspension and floc matrix were likely
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a dynamic double-layered EPS structure that contained loosely bound EPS (LB-EPS)
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and tightly bound EPS (TB-EPS) [24,25]. Fig. 5 a) showed the changes of EPS under different concentration of Mg2+. LB-EPS and TB-EPS were slight changed when Mg2+
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concentration increased from 0.45 to 1.1 mmol/L. LB-EPS was around 43.6 mg TOC/g VSS and TB-EPS was 45.6 mg TOC/g VSS. When Mg2+ concentration increased to 3
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mmol/L, EPS greatly reduced. LB-EPS and TB-EPS were respectively reduced to 16.6
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and 32.7 mg TOC/g VSS. It's worth noting that the stratification components of EPS changed as TB-EPS was much higher than LB-EPS. Previous studies verified that the
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ratio of LB-EPS/EPS was related to sludge flocculability and dewaterability. Particularly, LB-EPS was closely correlated with the bioflocculability and excessive LB-EPS deteriorated the sludge flocculability [26,20]. In our study, LB-EPS/EPS decreased from 49.5% to 33.6% when Mg2+ concentration increased from 0.45 to 1.1 mmol/L. FA of sludge increased and SVI declined. The decrease of LB-EPS/EPS is the
12
main reason for the improvement of sludge flocculation. Fig. 5. Effects of Mg2+ on EPS, PN and PS: a) EPS, b) PN and PS of LB-EPS, c) PN and PS of TB-EPS.
PN and PS containing a large quantity of functional groups were the major components of EPS and they had a great impact on the properties of microbial aggregates [27,28]. As shown in Fig. 5 b) and c), PN and PS slightly increased when
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Mg2+ concentration increased from 0.45 to 0.65 mmol/L because it might promote the
synthesis of nucleic acids and proteins. PN declined when Mg2+ concentration increased from 0.65 to 3 mmol/L. However, Mg2+ had a slightly different effect on the content of
-p
PS. PS content in the TB-EPS did not significantly change, but PS content in the LB-
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EPS had a same variation tendency as PN and evidently declined under Mg2+ concentration of 3 mmol/L. Higher concentration of Mg2+ might suppress the
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production of PS, which was consistent with the finding of Sajjad and Kim [29].
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3.3.2. EEM fluorescence spectra of EPS at different Mg2+ concentration EEM fluorescence spectra of LB-EPS and TB-EPS under 0.45, 1.1 and 3 mmol/L
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of Mg2+ concentration were shown in Fig. 6. Each EEM fluorescence spectrum
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provided special information about the chemical composition of EPS. Four dominant peaks could be identified according to the method described by Chen et al. [30]. Peak A in LB-EPS and TB-EPS was located at the excitation/emission wavelengths (Ex/Em) of 255-265/315-330nm, and Peak B was situated at 220-230/325-335nm. Peak A and Peak B were assigned to tryptophan-like substances and tyrosine-like substances, respectively. Peak C in TB-EPS was identified at Ex/Em of 245-260/420-440nm, 13
corresponding to humic acid-like substances. Peak D was identified at Ex/Em of 290325/370-440nm, which was corresponded to humic acid-like substances. The tryptophan-like (Peak A) and tyrosine-like substances (Peak B) were two major substances in LB-EPS and TB-EPS. In addition, humic acid-like substances (Peak C) existed in TB-EPS was stayed at a relatively low content. The intensity of peak A and peak B increased with the increase of Mg2+ concentration, indicating that the aromatic
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protein in EPS increased. The flocculation capability of sludge improved with the
increase of aromatic protein because the aromatic protein-like substance might play an important role in maintaining the stable structure of the sludge flocs [12,31].
-p
Fig. 6. EEM of LB-EPS and TB-EPS under different concentration of Mg2+: a1) LB-EPS at 0.45
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mmol/L of Mg2+, b1) LB-EPS at 1.1 mmol/L of Mg2+, c1) LB-EPS at 3 mmol/L of Mg2+, a2) TBEPS at 0.45 mmol/L of Mg2+, b2) TB-EPS at 1.1 mmol/L of Mg2+, c2) TB-EPS at 3 mmol/L of Mg2+.
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Additionally, all the fluorescence peaks either shifted towards longer (red shift) or
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shorter wavelengths (blue shift) according to the emission and/or excitation scale. Compared with the fluorescence peak location of LB-EPS and TB-EPS at 0.45 mmol/L
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of Mg2+, Peak A in the TB-EPS was red-shifted by 10 nm along the Ex axis under 1.1 and 3 mmol/L of Mg2+, which might be attributed to the increase of carbonyl-containing
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substituents, hydroxyl, amino groups and carboxyl constituents [32]. Peak B in the LBEPS was blue-shifted by 5 nm along the Em axis under 3 mmol/L of Mg2+, and that in the TB-EPS was blue-shifted 10 nm along the Ex axis and red-shifted by 5 nm along the Em axis under 1.1 mmol/L of Mg2+. The shift of Peak B could be associated with the reduction of some functional groups, such as aromatic ring and conjugated bond in 14
chain structures [33]. All the shifts of the fluorescence peaks could provide information on the changes of chemical structure, which would indicate the functional group change of EPS under different Mg2+ concentration. A significant increase of FA was observed under 1.1 and 3 mmol/L of Mg2+ concentration, which indicated that an increase of hydroxyl, amino and carboxyl constituents favored the bioflocculation.
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3.3.3. XPS analysis of EPS at different Mg2+ concentration Fig. 7 a) showed the X-ray photoelectron spectra of EPS obtained in the energy range of 0-1200 eV (1.0 eV step-size). Both LB-EPS and TB-EPS comprised similar
-p
elements under different Mg2+ concentration. The main elements were C, N and O that participated in the adsorption of Mg2+.
re
The changes of C 1s and O 1s functionalities in the LB-EPS and TB-EPS under
lP
different Mg2+ concentration were investigated to obtain more detailed information on the chemical bonding states of the elements (0.05 eV step size). The resulting peaks
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were later resolved into their individual components. High resolution spectra were shown in Fig. 7 b-e) only under 0.45 mmol/L of Mg2+ since XPS spectra were large
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homologous in terms of functionalities of EPS. Consequently, peak decomposition
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looked very similar and the chemical functional groups (molar ratio with respect to total carbon) were listed in Table 1. Fig. 7. a) XPS wide survey scans of LB-EPS and TB-EPS under different concentration of Mg2+
and XPS narrowly scanned spectra of b) C 1s of LB-EPS, c) C 1s of TB-EPS, d) O 1s of LB-EPS and e) O 1s of LB-EPS under 0.45 mmol/L of Mg2+.
The C 1s peak in both LB-EPS and TB-EPS was resolved into four component 15
peaks: (1) 284.8 eV C-(C,H) from hydrocarbons; (2) 286.2 eV C-(O,N) from proteins and alcohols; (3) 287.8 eV C=O or O-C-O from carboxylate, carbonyl, amide, acetals or hemiacetals; (4) 288.7 eV O=C-OR from uronic acids. The O 1s peak was decomposed into two peaks at 531.3 eV (O=C from carboxylate, carbonyl, ester, or amide) and 532.7 eV (O-(C,H) from hydroxide, acetals or hemiacetals). Only nonprotonated nitrogen compounds were detected in EPS, which indicated that amides
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(peptides) were dominant [34]. The peak pertaining to C=O was more prevalent in TBEPS (as shown in Fig. 8 and Table 1). However, the second O 1s component located at 531.3 eV presented an opposite situation. The binding energy of the two kinds of O was
-p
unchanged with the variation of Mg2+ concentration, which indicated that their
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chemical environment was similar and they did not participate in the chemical adsorption of Mg2+. An apparent trend could be observed in Table 2 that carboxylate
lP
(O531.3) contents of EPS increased with the addition of Mg2+ due to decreasing
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contribution from hemiacetals, acetals and alcohols (O532.7). C=O groups were often found in protein secondary structures and certain protein secondary structures promoted
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bioflocculation [35]. In our study, the proportions of carboxylate (O531.3) increased in EPS, which might the reasons for the enhancement of bioflocculation.
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Table 1. Functional group compositions obtained from narrowly scanned spectra of XPS.
3.4. Floc characteristics analysis of the activated sludge SVI and FA could characterize the settleability and flocculation of the sludge. The sludge growth was explored by measuring the sludge particle size. Fig. 8 a) showed that the average particle size of the sludge increased gradually with the increase of Mg2+ 16
concentration. When Mg2+ concentration increased from 0.45 to 3 mmol/L, FA increased from 15.4% to 29.2% and SVI decreased from 52.8 to 35.2 ml/g. The flocculation and settleability of activated sludge flocs were obviously improved with the increase of Mg2+ concentration. At the same time, the increase of Mg2+ concentration resulted in the change of sludge morphology from loose structured flocculent sludge to large and dense sludge particles (Fig. 8 b), c)). The ratios of
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extracellular PN/PS were related to the characteristics of bioflocs and biogranules
[36,37]. Aerobic bioflocs with a higher PN/PS ratio had a poor settleability [38,39]. In our work, PN/PS dropped from 3.5 to 1.2 when Mg2+ concentration increased from 0.45
-p
to 3 mmol/L (Section 2.3.1). SVI was in the range of 34.1-52.8 ml/g, which seemed to
re
be lower than that reported by Tay et al. [37]. It was because there was another potential factor affecting the settleability because Mg2+ increased the inorganic composition of
lP
floc. With the continuous increase of Mg2+ concentration, sludge flocs became denser
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and more compact [40]. Sludge flocs with poor settleability was washed out in the initial period due to short settling time, and then resulted in a low SVI value.
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Fig. 8. Effects of Mg2+ on the properties and morphology of sludge: a) sludge properties, b) sludge
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morphology at 0.45 mmol/L of Mg2+ (objective×40), c) sludge morphology at 3 mmol/L of Mg2+ (objective×40).
According to 3.2 and 3.3, the increase of Mg2+ concentration in the pellet was more
obvious than that in EPS. Both EEM and XPS results showed that the changes of Mg2+ concentration did not change the components and functional groups of EPS, but changed their proportions. Thus, Mg2+ concentration affected the flocculation and 17
settleablilty of sludge by changing the content of protein and polysaccharide in the EPS. Furthermore, the addition of Mg2+ increased the density of the flocs and the maximum SVI declined. The constant content of Mg2+ in the supernatant indicated that the neutralization of negative charge was not the main reason for the increase of bioflocculation.
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4. Conclusions The effects of Mg2+ concentration on the nitrifying activity, EPS and floc
characteristics of the activated sludge were investigated and the conclusions were as
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follows:
(1) The nitrification activity of the activated sludge promoted under low
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concentrations of Mg2+, while reduced under higher concentrations of Mg2+. When
lP
Mg2+ concentration was 1.1 mmol/L, the reactor had the optimal pollutant removal capacity and nitrification activity. Meanwhile, the sludge had good bioflocculation and
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sedimentation.
(2) The order of Mg2+ concentration in the activated sludge was as follows:
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pellet>supernatant>LB-EPS>TB-EPS. Mg2+ concentration in supernatant, LB-EPS and
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TB-EPS changed slightly, whereas significant changes were observed in the pellet. (3) High concentration of Mg2+ (3 mmol/L) reduced the EPS concentration and
changed its stratification components, while improved the bioflocculation and sedimentation performance of sludge and increased sludge particle size. The decrease of LB-EPS was the main reason for the improvement of bioflocculation. The increase of inorganic components and sludge particle size contributed to the enhancement of 18
settleability of the activated sludge. (4) The increase of Mg2+ concentration had slight influences on the type of fluorophores, but changed obviously their contents. The most components of LB-EPS and TB-EPS were similar and aromatic PN-like substances and humic acid-like substances were identified. The position of the fluorescent peak shifts and the chemical structure of EPS changed accordingly.
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Acknowledgements
The authors thank to the National Natural Science Foundation of China (grant
of
Jilin
Province
(grant
numbers
-p
numbers 51678119 and 51808254), the Science and Technology Development Program 20180201016SF,
20180101079JC
and
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20180101309JC) and the Science and Technology Research Project from Jilin
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Provincial Department of Education (grant numbers JJKH20180453KJ and JJKH20180454KJ) for their financial supports.
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[37] J.H. Tay, Q.S. Liu, Y. Liu, The role of cellular polysaccharides in the formation and stability of aerobic granules, Letters in Applied Microbiology 33 (2010) 222-226.
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[38] D.J. Batstone, J. Keller, Variation of bulk properties of anaerobic granules with wastewater type, Water Research 35 (2001) 1723-1729.
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[39] F. Mart í nez, J. Lema, R. M é ndez, F. Cuervo-L ó pez, J. G ó mez, Role of exopolymeric protein on the settleability of nitrifying sludges, Bioresource Technology 94 (2004) 43-48. [40] X.M. Li, Q.Q. Liu, Q. Yang, L. Guo, G.M. Zeng, J.M. Hu, W. Zheng, Enhanced aerobic sludge granulation in sequencing batch reactor by Mg2+ augmentation, 24
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Fig. 1. Schematic diagram of SBR. Fig. 2. Performances of nitrogen and COD removal. Fig. 3. Effects of Mg2+ on the nitrification activity of sludge. Fig. 4. Cations distribution in different EPS fractions. Fig. 5. Effects of Mg2+ on EPS, PN and PS: a) EPS, b) PN and PS of LB-EPS, c) PN and PS of TB-EPS.
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Fig. 6. EEM of LB-EPS and TB-EPS under different concentration of Mg2+: a1) LBEPS at 0.45 mmol/L of Mg2+, b1) LB-EPS at 1.1 mmol/L of Mg2+, c1) LB-EPS at 3
mmol/L of Mg2+, a2) TB-EPS at 0.45 mmol/L of Mg2+, b2) TB-EPS at 1.1 mmol/L of
-p
Mg2+, c2) TB-EPS at 3 mmol/L of Mg2+.
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Fig. 7. a) XPS wide survey scans of LB-EPS and TB-EPS under different concentration of Mg2+ and XPS narrowly scanned spectra of b) C 1s of LB-EPS, c) C 1s of TB-EPS,
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d) O 1s of LB-EPS and e) O 1s of LB-EPS under 0.45 mmol/L of Mg2+.
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Table 1. Functional group compositions obtained from narrowly scanned spectra of XPS.
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Fig. 8. Effects of Mg2+ on the properties and morphology of sludge: a) sludge properties, b) sludge morphology at 0.45 mmol/L of Mg2+ (objective×40), c) sludge morphology
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at 3 mmol/L of Mg2+ (objective×40).
26
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Figures and Tables
1. Electromagnetic stirrer 2. Rotameter 3. Air pump 4. Influent pump 5. Air diffuser 6. Drain valve 7. Sample port 8. Dissolved oxygen meter 9. pH meter
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lP
re
-p
Fig.1. Schematic diagram of SBR.
27
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Fig. 2. Performances of nitrogen and COD removal.
28
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na
lP
re
-p
Fig. 3. Effects of Mg2+ on the nitrification activity of sludge.
29
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na
lP
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-p
Fig. 4. Mg2+ distribution in pellet, TB-EPS, LB-EPS and supernatant.
30
Fig. 5. Effects of Mg2+ on EPS, PN and PS: a) EPS, b) PN and PS of LB-EPS, c) PN and PS of TB-
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ur
na
lP
re
-p
ro of
EPS.
31
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Fig. 6. EEM of LB-EPS and TB-EPS under different concentration of Mg2+: a1) LB-EPS at 0.45 mmol/L of Mg2+, b1) LB-EPS at 1.1 mmol/L of Mg2+, c1) LB-EPS at 3 mmol/L of Mg2+, a2) TB-
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EPS at 0.45 mmol/L of Mg2+, b2) TB-EPS at 1.1 mmol/L of Mg2+, c2) TB-EPS at 3 mmol/L of Mg2+.
32
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Fig. 7. a) XPS wide survey scans of LB-EPS and TB-EPS under different concentration of Mg2+
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and XPS narrowly scanned spectra of b) C 1s of LB-EPS, c) C 1s of TB-EPS, d) O 1s of LB-EPS
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and e) O 1s of LB-EPS under 0.45 mmol/L of Mg2+.
33
Table 1. Functional group compositions obtained from narrowly scanned spectra of XPS. Element
Atomic (%)
Peak (eV)
Atomic (%)
Assignments
a1
a2
a3
b1
b2
b3
284.8
82.70
61.08
66.21
58.23
65.50
58.05
C-(C,H)
286.2
7.77
31.95
23.47
22.79
22.96
20.62
C-(O,N)
287.8
5.00
0.40
3.83
17.7
2.88
15.41
C=O+O-C-O
288.7
4.46
6.57
6.49
1.27
8.66
2.48
O=C-OH
531.3
11.50
34.75
48.27
28.22
41.78
69.78
O=C
532.7
88.49
65.24
51.73
71.78
58.82
30.32
C-OH; C-O-C
C 1s
O 1s
Notes: a1, a2 and a3 corresponding to LB-EPS at Mg2+ concentration of 0.45, 1.1 and 3 mmol/L,
ro of
respectively. b1, b2 and b3 corresponding to TB-EPS at Mg2+ concentration of 0.45, 1.1 and 3
Jo
ur
na
lP
re
-p
mmol/L, respectively.
34
ro of -p re lP
Fig. 8. Effects of Mg2+ on the properties and morphology of sludge: a) sludge properties, b) sludge
Jo
ur
(objective×40).
na
morphology at 0.45 mmol/L of Mg2+ (objective×40), c) sludge morphology at 3 mmol/L of Mg2+
35