Pre-acidification greatly improved granules physicochemical properties and operational stability of Upflow anaerobic sludge Blanket (UASB) reactor treating low-strength starch wastewater

Journal Pre-proofs Pre-acidification greatly improved granules physicochemical properties and operational stability of Upflow Anaerobic Sludge Blanket (UASB) reactor treating low-strength starch wastewater Jiang Wu, Bo Jiang, Bo Feng, Lu Li, Siti Nur Fatihah Moideen, Hong Chen, Chaimaa Mribet, Yu-You Li PII: DOI: Reference:

S0960-8524(20)30079-1 https://doi.org/10.1016/j.biortech.2020.122810 BITE 122810

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Bioresource Technology

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28 June 2019 10 January 2020 13 January 2020

Please cite this article as: Wu, J., Jiang, B., Feng, B., Li, L., Nur Fatihah Moideen, S., Chen, H., Mribet, C., Li, YY., Pre-acidification greatly improved granules physicochemical properties and operational stability of Upflow Anaerobic Sludge Blanket (UASB) reactor treating low-strength starch wastewater, Bioresource Technology (2020), doi: https://doi.org/10.1016/j.biortech.2020.122810

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Pre-acidification greatly improved granules physicochemical

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properties and operational stability of Upflow Anaerobic

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Sludge Blanket (UASB) reactor treating low-strength starch

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wastewater

5 6

Jiang Wu a, Bo Jiang a, Bo Feng b, Lu Li a, Siti Nur Fatihah Moideen b, Hong Chen c,

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Chaimaa Mribet b, Yu-You Li a,b*

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a

Department of Civil and Environmental Engineering, Graduate School of

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Engineering, Tohoku University, 6-6-06 Aza-Aoba, Aramaki, Aoba Ward, Sendai,

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Miyagi 980-8579, Japan.

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b

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Aramaki, Aoba Ward, Sendai, Miyagi, 980-8579, Japan

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c

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Hunan Province, School of Hydraulic Engineering, Changsha University of Science

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&Technology, Changsha 410004, China

Graduate School of Environmental Studies, Tohoku University, 6-6-06 Aza-Aoba,

Key Laboratory of Water-Sediment Sciences and Water Disaster Prevention of

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*Corresponding author: Yu-You Li

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E-mail: [email protected] 1

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Abstract A two-stage process consisting of a pre-acidification unit and an Upflow Anaerobic

22

Sludge Blanket (UASB) reactor (UASBT-S) was compared with a one-stage UASB

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reactor (UASBO-S) to evaluate the treatment stability of starch wastewater (SW). The

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Two-stage process provided higher treatment stability than UASBO-S. Sludge

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floatation occurred in the UASBO-S when the organic loading rate (OLR) was

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increased to 4g-COD/L/d, beyond which a paste-like membrane structure adhered to

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the granules was observed. Further analysis suggests that the substrate derived

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polysaccharide components embedded in the loosely-bound extracellular polymeric

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substances (LB-EPS), triggered significant increase in the protein/polysaccharide ratio

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in the tightly-bound EPS (TB-EPS), and was suggested to result in the granules

31

floatation and disintegration. During the pre-acidification, the starch was mainly

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converted to acetic and propionic acids. The pre-acidification was beneficial for

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reducing the EPS content fluctuations in the UASBT-S, which greatly improved

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settling capability and strength of the granular sludge.

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Keywords

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Starch wastewater; UASB reactor; Pre-acidification; Bound EPS; Granular sludge

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floatation

42 43

1. Introduction

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Starch is widely used in the paper, cardboard, and corrugation industries

45

(Nikodinovic-Runic et al., 2013). With the total starch consumption in 2011 at

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approximately 70 million tons, world-wide starch production maintains an upward

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trend (Nikodinovic-Runic et al., 2013). In China, the world’s largest producers of

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sweet potato, around 1.5 million ton of sweet potato was used in starch production in

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2011 (Xu et al., 2017). Notably, the starch residue is discarded at many points in the

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production chain. For example, during the roughing process, in excess of 10 m3 of

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starch wastewater (SW) is typically discharged in the processing of one ton of sweet

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potato root (Xu et al., 2017). One type of SW discharged from the cornstarch

53

production plant, for instance, had a high chemical oxygen demand (COD) of 40,000-

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50,000 mg/L, and pH of 3.0-5.0 (Xue et al., 2010). Such a large-scale waste streams,

55

once large-scale discharged into natural waters without any treatment, can lead to a

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range of ecological problems.

3

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Awareness of the environmental hazards of waste streams has led to the

58

development of various treatment strategies. Among the alternatives for organic waste

59

streams treatment strategies, anaerobic digestion (AD) is a biological process which

60

converts organic compounds into different products under the oxygen-free condition.

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Biogas produced by AD process contains 50-75% methane and 5-10% hydrogen, and

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is a potential biofuel (Kamali et al., 2016; Wu et al., 2019). Starch belongs to

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carbohydrates, and is a linear polysaccharide (PS) with α‐(1‐4)‐linked D‐glucose

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units. In nature, it is used primarily for energy storage by plants and biological cells

65

(Li et al., 2010; Pérez and Bertoft, 2010). SW is non-toxic, with relatively high

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BOD/COD ratio of 0.4-0.7, and is biodegradable (Mutamim et al., 2013; Sun et al.,

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2012; Xue et al., 2010). The Up-flow Anaerobic Sludge Blanket (UASB) reactor, one

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of the most effective AD processes, has been attracted much of research attention in

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the last several decades (Wu et al., 2018). Much of the focus of investigations into the

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UASB treatment has been on shortening the hydraulic retention time (HRT) to

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observe the organics removal and biogas production under the elevated organic

72

loading rate (OLR) (Kong et al., 2019). Parawira et al. (2006) increased OLR from

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1.5 to 7g-COD/L/d in UASB treatment of the potato leachate by decreasing HRT

74

from 13.2 to 2.8 days, verified that the COD removal efficiency was 93±5.3% when

75

the OLR reached 6.1g-COD/L/d. Sun et al. (2012) employed a modified UASB 4

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reactor to treat SW and found that the maximum methane production rate was up to

77

0.55L-methane/L-effective volume/day (this unit is represented as L/L/d below) under

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an OLR of 16g-COD/L/d. These efforts indicate that UASB reactor is suitable for

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stable organic conversion in SW treatment under the lower OLR conditions.

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However, Lu et al. (2015) employed a UASB reactor that treated low-strength SW,

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and noted that the emerged operation risks of sludge floatation, washout and

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disintegration, were ascribed to the advantageous structure of the extracellular

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polymeric substance (EPS) in the granular sludge that destroyed when the OLR was

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increased to 6g-COD/L/d. This indicated that besides the focuses on the process

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organics removal performance, the unstable physicochemical properties of granular

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sludge when using UASB reactor to treat complex wastewaters (such as the SW), is

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also a critical issue that cannot be bypassed.

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The organics conversion of carbohydrates in the AD process broadly includes two

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steps, acidogenensis and methanogenesis (Yang et al., 2003). The biokinetic and

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physiologic characteristics of the related-microorganisms of the two steps are

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different. The pre-acidification is the primary step of the two-stage AD process, is

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employed to separate the acidogenesis from methanogenesis under ex-situ condition,

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and could help to ease the rate-limitation of methanogenesis in the bioreactors. The

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complex organic compounds are degraded to small molecule organics via different 5

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metabolic pathways in the pre-acidification unit, such as butyric, propionic and acetic

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acids, which are all classified as volatile fatty acids (VFAs). It is reported that the

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UASB could perform excellent treatment capability for the VFAs, including

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considerable organics conversion feasibility, as well as satisfactory granule

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microstructure and settling properties (Fang et al., 1995 (a); Fang et al., 1995 (b);

100

101

Wong et al., 2008).

Recent few studies have shown that the pre-acidification successfully improved

102

anaerobic treatment performance of waste streams, such as brewery wastes (Ahn et

103

al., 2001), cheese whey (Diamantis et al., 2014) and chicken manure (Çalışkan and

104

Azbar, 2017). Unfortunately, to date, there is little information available regarding the

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insight into the gradual deterioration of granular sludge in the UASB treatment of SW

106

and how the pre-acidification makes possible impacts on stability of granular sludge

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in the UASB reactor. Thus during this work, a one-stage UASB reactor and a pre-

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acidification unit introduced in a Two-stage UASB process were operated for 280

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days, with low-strength SW feeding. The objectives were: 1) to compare the overall

110

treatment performance in both processes in terms of organics removal capability, the

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physical characteristics of granular sludge (e.g. diameter size distribution, surface

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structure and granules settling capability) as well as operational stability; 2) to

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evaluate chemical properties of granular sludge in both processes based on elemental 6

114

and spectroscopic analysis of sludge components and chemical compositions of the

115

bound EPS, in order to give insights into the gradual deterioration of granules; 3)

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finally to find the optimal operational conditions in the UASB treatment of SW.

117 118

2. Materials and methods

119

2.1 Lab-scale reactors and the operational conditions

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A schematic diagram of the reactor setup including the accessories is provided in

121

Fig.1. The apparatus used in the long-term experiment comprised a One-stage UASB

122

reactor (UASBO-S) and a Two-stage UASB reactor (UASBT-S). The working volume

123

of both UASB reactors was 6L with a vertical height of 0.8m. A CSTR reactor

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(CSTRT-S) with an effective volume of 6L was employed as the pre-acidification unit

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in the Two-stage process (T-S process). Water bath heaters (NTT-20S, EYELA) were

126

equipped to maintain the reactors at a mesophilic condition (35±1℃). A plastic tank

127

with an effective volume of 200L was employed to store the substrate. A condenser

128

(Yamato, BE201) was inserted into the plastic tank to refrigerate the substrate at 4°C.

129

Peristaltic pumps (Masterflex, EW-07528-10) were used to continuously feed the

130

substrate. Gas flow meters (SHINAGAWA W-NK-0.5) were used to measure the

131

daily biogas production. In this study, the OLR of both processes was increased by

7

132

shortening the HRT. The detailed operational conditions of the long-term experiment

133

are shown in Table 1.

134 135

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2.2 Wastewater and seed sludge inoculation

The SW contained 1000mg/L of COD from soluble starch. Supplements of the

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essential elements were as follows (mg/L): NH4Cl (1000), KCl (750), K2HPO4 (250),

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MgCl2·6H2O (125), KH2PO4 (100), FeCl2·4H2O (42), Na2SO4 (30), CaCl2·2H2O (15),

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NiCl2·6H2O (4.2) and CoCl2·6H2O (4.2). The phased quality parameters of the

140

substrate are shown in Table 2.

141

Seed granular sludge was withdrawn from a full-scale mesophilic UASB reactor in

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a brewery wastewater treatment plant, Japan. 5.0 L of the substrate was mixed with

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1.0 L of digested sludge taken from a domestic wastewater treatment plant in Sendai,

144

Japan, then was inoculated into the CSTRT-S. Hydrochloric acid (HCl) was used to

145

adjust the initial pH of this mixture sludge to 5.5, the appropriate value for acidogens

146

(Y. Wang et al., 2017).

147 148

149

2.3 Testing and analytical methods

A gas chromatograph (SHIMADZU GC-8A) equipped with a thermal conductivity

8

150

detector (SHINAGAWA W-NK-0.5) was employed to analyze biogas components

151

(N2, CH4, and CO2). The 2m length of the steel column of the GC was packed with

152

Porapak Q. Helium was the carrier gas. A pH meter (HM-30 R, DKK-TOA) was

153

utilized to measure the substrate, influent and effluent pH values. The VFAs were

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analyzed by a gas chromatograph equipped with a flame ionization detector (GC-FID,

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Agilent 6890N). A capillary column (30m, DB-WAXetr) was equipped with the GC-

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FID, using nitrogen as the carrier gas. The VFAs samples were filtered through a 0.45

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μm hydrophilic polyethersulfone membrane. The same filtration method was also

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used in the testing of soluble COD (SCOD), soluble carbohydrates, and protein (PN).

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Before the VFAs test, 0.5 ml of the filtered liquid sample was mixed with 0.5 ml of

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0.1M HCl to adjust the pH around 3.0.

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Elemental analysis and Fourier-transform infrared spectroscopy (FTIR) analysis of

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the sludge were conducted at the HRT of 6h with the OLRs of 6g-COD/L/d. All the

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samples were dried in an oven with 105℃ for 2 hours and been fully ground before

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testing. The elemental composition (C,H,N,O,S) of granular sludge was analyzed by

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an inductively coupled plasma mass spectrometry (ICP-MS, Thermo Fisher Scientific,

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iCAP6300). The infrared spectra of modified starch, granular sludge and the surface

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adhered structure stripped from the granular sludge at UASBO-S were scaned with an

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FTIR spectrometer (ThermoFisher, Nicolet6700). 9

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COD, carbohydrates (including the PS), PN, and mixed liquor volatile suspended

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solids (MLVSS) of the granular sludge followed the Standard Methods

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(APHA/AWWA/WEF, 2012). A vis spectrophotometer (HACH, DR5000) was

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applied in the spectrophotometry. The morphologic properties of granular sludge

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sampled from the UASB reactors were characterized by scanning electron microscopy

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(SEM, HITACHI, S-3000N). A statistical software (SPSS®, IBM, USA) was used for

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statistical hypothesis tests.

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2.4 Particle size distribution and bound EPS analysis

Granular sludge was sampled from the UASBO-S and UASBT-S in the last 1-5 days

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of each OLR condition, to determine the change in the diameter size distributions and

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the EPS components. Sludge samples were collected from 15, 45, 64cm of the reactor

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height, referred to the "Top", "Middle" and "Bottom" of UASB reactors. Six screen

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meshes with different pore sizes (0.35mm, 0.5mm, 1.0mm, 2.0mm, 2.8mm, and

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4.0mm) were used to filter and classify the granular sludge by diameter size, and

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converted the size distributions to percentages (total granules >1000). An image

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analyzing software (Image J, National Institutes of Health, USA) was used for

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granules counting. Gaussian distribution model (Eq.(1)) was selected to fit the curve

10

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of diameter size distributions of granular sludge. 2

188

189

𝑦 = 𝑦0 + w

A

― (x ― xc)

ⅇ

2w2

2π

Eq.(1)

Where y0 is the initial distribution frequency; A is a constant, which represents

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maximum distribution frequency (%); w is the variance; Xc is the mathematical

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expectations of the median size of the granules (mm), corresponding to the highest

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granular size distribution.

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Extraction of the loosely-bound EPS (LB-EPS) and tightly-bound EPS (TB-EPS)

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were carried out with the cationic exchange resin (CER) method, mainly based on

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Zhang et al. (2016). The variations in the PN and PS contents in the bound EPS were

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determined in this study. A three-dimensional excitation-emission matrix (3D-EEM,

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HITACHI, F-7000) fluorescence spectra were applied to record the intensities of

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protein-like and humic acid-like substances contained in the bound EPS. 3D-EEM

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fluorescence spectra were collected with subsequent scanning emission spectra (Em)

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from 220 to 450 nm at 5 nm increments by varying the excitation (Ex) wavelength

201

from 250 to 600 nm at 5 nm increments. The photomal voltage was set at 400V.

202 203

3. Results and discussion

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3.1 Comparison of the processing performance of the One-stage (O-S) and Two-stage 11

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(T-S) processes

In order to make the comparisons intuitive, the long-term records for each

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parameter were reorganized and listed according to ascending OLR of 1, 2, 4 and 8g-

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COD/L/d, the operational conditions covered in both processes. Fig. 2 (a) and (b)

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present the SCOD removal efficiencies of the O-S and T-S processes: the stepwise

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increased OLR did not result in the perceptible fluctuation of effluent SCOD

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concentrations in both processes: the overall SCOD removal efficiencies in both

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processes were maintained at 85% or higher, irrespective of the OLR settings. It is

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worth mentioning that the effluent SCOD in the CSTRT-S was reduced by 41.4%-

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55.2% from the influent. Similar phenomenon was also reported by Yu et al. (2016),

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who noted that 23.5% of the COD loss which occurred in an unsterilized complex

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pre-acidification unit in the treatment of corn stalks and cow dung waste, was due to

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the organic nutrient consumption of microorganisms (including acidified bacteria and

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some miscellaneous microorganisms) growth. Meanwhile, Akutsu et al. (2009)

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pointed out that proceeded methanogenesis and hydrogenesis in the pre-acidification

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unit were potential causes of the COD loss. In fact, on one hand, the dosage of

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moderate amount of sodium bicarbonate in the substrate contributed to alkalinity

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maintenance, since the pH in both UASB reactors were maintained around 7.0, as

223

shown in Fig.2 (c) and (d), which is the ideal value for methanogens (Ahmad et al., 12

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2011). On the other hand, the pH values in the CSTRT-S that ranged from 6.4 to 6.8,

225

were insufficient to totally suppress metabolic activities of methanogens (W. Zhang et

226

al., 2019). This probably resulted in the COD loss in the CSTRT-S. In the subsequent

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experiments, a strategy was adopted that reduced the alkalinity supply to the substrate

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for decreasing pH of the CSTRT-S to around 5.0, by pumping an acid-base buffer (a

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mixture of sodium bicarbonate and caustic soda) to the UASBT-S to maintain the pH

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value higher than 7.0. This increased 20-25% of the COD concentration in the

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CSTRT-S effluent and increased 10-15% of methane production in the follow-up

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UASBT-S (data not shown).

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As shown in Fig. 2 (e), the soluble carbohydrates removal efficiencies (CREs) in

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the UASBO-S closed to 95% at an OLR range of 1-4g-COD/L/d. This is clear evidence

235

that excellent starch conversion could be achieved in a UASB reactor with a lower

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OLR, which has been agreed upon with the report by Lu et al. (2015), who obtained a

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satisfactory UASB treatment performance of SW at an OLR of 4g-COD/L/d. It was

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obvious that in the UASBO-S, the CRE at the OLR of 8g-COD/L/d was significantly

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lower than other experimental periods (one-tailed t-test, the p-value <0.05), and has

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decreased to 88.8%. In the methanogenesis, In-situ acidification procedure of

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converting carbohydrates to short-chain fatty acids is fast, but should not to be

242

considered as a rate-limiting step (Ma et al., 2018). At the OLR of 8g-COD/L/d, as 13

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shown in Fig.2 (g), only 30mg/L of acetic acid was detected in the UASBO-S effluent,

244

without significant VFAs accumulation. This suggests that the decrease of

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carbohydrates conversion in the UASBO-S at the OLR of 8g-COD/L/d was not

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contributed to residual starch in the effluent. As can be seen in Fig.2 (i), the MLVSS

247

of granular sludge in the UASBO-S started to show a downward trend at an OLR of

248

4g-COD/L/d, and further decreased to 47.54g/L when the OLR was increased to 8g-

249

COD/L/d. It is known that the MLVSS of the sludge is positively correlated with the

250

biomass amount in the bioreactor (F. Zhang et al., 2019). This implied that when the

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OLR was increased to 4g-COD/L/d, the granules disintegration resulted in the

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biomass loss in the UASBO-S. Furthermore, at the OLR of 4g-COD/L/d, the granules

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settling velocities (SV) in the UASBO-S performed lower than that in the UASBT-S.

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This could also reflect the biomass loss and the worsened sludge granulation in the

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UASBO-S (Ahmad et al., 2011). Hence, the decrease in the carbohydrate removal

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capability in the UASBO-S at the higher OLR conditions is suggested to attribute to the

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dissolution of intracellular PS components caused by the cytoplasmic release of

258

microorganisms. As shown in Fig.2 (f), 95% of carbohydrates could be removed in

259

the pre-acidification unit, the overall CRE could be maintained higher than 95%

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throughout the whole experiment. The improvement of the CRE in the T-S process,

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especially at higher OLR was apparently due to the creation of better acidification 14

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conditions, viz., the influent carbohydrates (starch) could be converted to acetic and

263

propionic acids during the pre-acidification, as shown in Fig.2 (h). In other words, the

264

UASBT-S was mainly fed with the acetic and propionic acids as the substrate. Because

265

of the biochemical conversion from propionate to acetate is thermodynamically

266

unfavorable (ΔG=+76.1 kJ/mol), in the AD process, the accumulation of propionate is

267

not usually conducive to methanogenesis (Feng et al., 2015; Li et al., 2018). However,

268

the concentrations of the propionic acid in the CSTRT-S were at the range of 88-

269

193mg-HAc/L (equivalent to 109-238mg/L of propionate), much lower than the

270

1600mg/L of propionate, one case of concentration threshold which inhibited the

271

mesophilic AD process reported by Li et al. (2018). On the other hand, Ahn et al.

272

(2001) advocated that the partial pre-acidification (20-50%) could reduce the risk of

273

granulation deterioration in the subsequent UASB reactor. As shown in Fig.2 (h) and

274

(i), neither VFAs accumulation nor decrease in the MLVSS of granular sludge was

275

observed in the UASBT-S. Although the complete pre-acidification in this study

276

showed no adverse impacts on both the sludge granulation and long-term UASB

277

treatment stability of SW, in the future work, extra attention should be paid to the

278

effects of acidification degree at the different HRT and even the size volume of the

279

pre-acidification unit on the treatment performance of SW, since it gives opportunities

280

to optimize the Two-stage process. 15

281

3.2 Temporal variation of the distributions in the diameter size of the granular sludge

282

The distributions in the size of the granules along the vertical of both UASBO-S and

283

UASBT-S are shown in Fig.3. The diameter size distributions along both UASB

284

reactors were generally consistent when the OLRs were set lower than 2g-COD/L/d,

285

and they well to the Gaussian distribution model (the correlation coefficients (R2)

286

were in the range of 0.8531-0.9999). Liu et al. (2011) applied an electric field to

287

enhance the granulation of anaerobic sludge in a UASB reactor, and observed the

288

similar nonlinear feature of the diameter size distributions in the healthy and mature

289

granular sludge. In this study, at the OLR of 2g-COD/L/d, the granules in both UASB

290

reactors showed compact granules structure with a smooth surface. During this OLR

291

setting, the vertical distribution in the median diameter size of the granules in the

292

Gaussian fitting, characterized as the mathematical expectation (XC), stepwise panned

293

right along the X-axis from 1.34 to 1.69mm and 1.36 to 1.75mm, corresponding to the

294

median size of granules along the UASBT-S and UASBO-S from the "Top" to

295

"Bottom". This suggests that the larger granules with larger density that made them

296

perform a higher settleability. The larger granular sludge within a certain diameter

297

range suggests a higher microbiological activity, as reported by Bhunia and

298

Ghangrekar. (2007), who found that at a diameter range of 0.3-3.0mm, the

299

methanogenic capacity of anaerobic granules was enhanced as the diameter size 16

300

increased. This suggests that the granules in the bottom of UASB reactors showed

301

higher methanogenic activity.

302

After increasing the OLR of UASBO-S to 4g-COD/L/d, larger granules with diameter

303

size of 2.8-4.0mm started to be observed at both "Top" and "Bottom" part of the

304

reactor, as shown in Fig.3, larger granules gathered to the "Top" (and "Bottom") part

305

when increased the OLR to 6-8g-COD/L/d (see the green bars). none of the granules

306

were in the "Middle" of the UASBT-S at the OLRs of 4-8g-COD/L/d since the sludge

307

stratification. On the other hand, a sharp increase in the smaller granules with

308

diameter size of 0.35-0.5mm was observed in both of upper and lower ends of the

309

UASBO-S since increasing the OLR to 4g-COD/L/d (see the blue bars). During these

310

periods, the diameter size distributions of granules in the UASBO-S was clearly

311

polarized, statistically disobeying the Gaussian distribution. All these suggest that

312

sludge deterioration (granules agglomeration or disintegration) could have occurred in

313

the UASBO-S under the higher OLR settings. From the perspective of the granules

314

diameter size distribution, no significant changes were noted in the UASBT-S with the

315

increase in OLR.

316

17

317

318

3.3 Physicochemical properties of the granular sludge

As shown in Fig.4 (a), the floatation of granules started to be observed in the

319

UASBO-S when the OLR was increased to 4g-COD/L/d with the shortening of the

320

HRT to 6h. The floatation became aggravated when the OLR was further increased.

321

At the OLR of 4g-COD/L/d, in the UASBO-S, a paste-like membrane structure was

322

observed on the surface of the granules cultivated, visible to the naked eye (Fig.4 (c)

323

and (e)). SEM micrographs obviously showed that a ribbon-like attachment covered

324

the surface of the granules sampled from the UASBO-S. The similar phenomenon was

325

documented by Lu et al. (2015), who observed a claw-like attachment on the surface

326

of the anaerobic granules when fed with starch as the sole organic substrate. On the

327

other hand, as shown in Fig.4 (b), the settlement characteristics of granular sludge

328

remained relatively stable in the UASBT-S when the OLR was increased to 4g-

329

COD/L/d or higher. The granules in the UASBT-S was compact and showed the

330

spherical outer shape with a clear outline (Fig.5 (d) and (f)).

331

Gagliano et al. (2017) gave an insight into one type of long filamentous outgrowth

332

on the granular sludge in a UASB reactor, and pointed out that it had a similar

333

chemical composition to medium-chain fatty acids. According to Gagliano et al.

334

(2017), the long filamentous outgrow associated with the Methanosaeta

18

335

harundinacea, a methanogens species grown at a specific acetate concentration

336

threshold, could contribute to the sludge granulation and promote the strength of

337

granules by immobilizing other microorganisms. These descriptions are obviously

338

inconsistent with the low MLVSS (as mentioned in section 3.1), uneven diameter size

339

distributions (as mentioned in section 3.2), and the poor settling characteristics of the

340

granular sludge in the UASBO-S at higher OLR conditions. It is worth to mention that

341

the granules with a form of the flagella-like filament outgrowing on the granules was

342

commonly observed in the UASBT-S with an OLR of 8g-COD/L/d, and the granules

343

remained compact physical structure and outstanding settling capability. Thus far,

344

there are two forms of granules surface hyperplasia structure were observed in the two

345

UASB reactors respectively. The paste-like attachment on the surface of the granules

346

observed in the UASBO-S may not be related to the enrichment of Methanosaeta in the

347

granular sludge. The difference of microbial community distribution in the two UASB

348

reactors will not be discuessed in-depth in this work.

349

As shown in Fig.4 (g-h), elemental compositions analysis of granular sludge

350

sampled from the "Bottom" of both UASB reactors were analyzed at an OLR of 4g-

351

COD/L/d. The molecular formula of the granular sludge taken from the UASBO-S and

352

UASBT-S reactors was presumed to C5H9.78O3.97N0.98 and C5H9.39O3.57N0.88,

353

respectively. Both of them were similar to C5H9O3N, the molecular formula of 19

354

anaerobic cells (Chen et al., 2017). Subsequently, FTIR analysis of the two granules,

355

the paste-like membrane structure stripped from the granules in the UASBO-S and the

356

pure modified starch (the substrate) was carried out to identify their chemical groups.

357

The definition of vibrational assignments and functional substance for FTIR spectra

358

mainly based on the Wang et al. (2018) and Zhang et al. (2018). Comparing with the

359

chemical groups of the granules sampled from the UASBT-S, more stronger bands of

360

1626, 1538, 1231 and 1018cm-1 appeared in the FTIR spectra of the granules sampled

361

from the UASBO-S. These bands correspond to the amide-ralated groups (e.g., C=O,

362

N-H, -COO-, etc.) and C-O-C stretches, suggesting that granular sludge in the

363

UASBO-S with the high concentrations of the PN and PS components. A significant

364

band at 1405cm-1 was only recorded in the spectra of the granules in the UASBO-S.

365

The band corresponds to C=O stretches in the carboxyl, associated with the amino

366

groups (amide III). This suggests that the peptide hydrolysis occurred in the UASBO-S.

367

The FTIR spectra of the stripped membrane structure shows the coincident peak shape

368

with the granular sludge sampled from UASBO-S and UASBT-S reactors. This is

369

inconsistent with the peak shape recorded from modified starch, which exhibited a

370

deletion in protein-related functional groups.

371

Hence, it could be speculated that the essence of the paste-like membrane structure

372

in the UASBO-S is a form of the gelatinized biopolymers, firmly attached to granules. 20

373

In the UASBO-S, the paste-like membrane structure connecting the micronized

374

granules, exhibited a clear cementation effect. As shown in Fig.4 (e), the granules

375

aggregates showed a larger diameter size. These explained the polarization of the

376

granules diameter size distribution in the UASBO-S. In addition, the higher specific

377

surface of the granular aggregates could hinder the internal transfer of biogas bubbles

378

(Lu et al., 2015). Also, the biogas bubble is more likely to adhere to the paste-like

379

membrane structure, therefore reduce the specific gravity of granules (B. Wang et al.,

380

2017). This could explain why the granules floatation occurred in the UASBO-S. In

381

contrast, the change in the granules surface texture was effectively avoided in the

382

UASBT-S, which ensured the stable morphology structure and satisfactory settling

383

capacity of the granules.

384 385

386

3.4 Variation of EPS contents in the granular sludge

The EPS is a general term for a group of biopolymers either secreted by

387

microorganisms or generated via the cell adsorption and conversion of organic

388

components in the substrate. It has been shown that EPS significantly influence the

389

sludge properties, including settling capability, surface morphology and surface

390

charge (Sheng et al., 2010). Besides containing a small quantity of ingredients such as

21

391

humic acids, nucleic acids, etc. EPS is mainly composed of PN and PS, estimated at

392

more than 75% of the total EPS content (Basuvaraj et al., 2015). According to the

393

binding force of cells, bound EPS can be further subdivided into LB-EPS, depicted as

394

"loose and dispersible slime layer" and TB-EPS, depicted as "the inner layer which

395

tightly and stably bound to the cell surface" (Sheng et al., 2010; Zhang et al., 2016; B.

396

Wang et al., 2017). In this section, the bound EPS was extracted from the granular

397

sludge collected from the "Bottom" of the UASB reactors. The variations of LB-EPS

398

and TB-EPS in the two UASB reactors are shown in Fig.5 (a) and (b), respectively.

399

As shown in Fig.5 (a), the LB-EPS in the UASBO-S (the left bars of side-by-side bar

400

charts) varied significantly (p-value <0.05) with increasing in OLR: as the OLR

401

increased from 1 to 8g-COD/L/d, the LB-EPS sharply increased from 4.4 to 37.0mg-

402

EPS/g-VSS. Correspondingly, as shown in Fig.5 (b), the TB-EPS in the UASBO-S

403

increased from 62.0 to 223.8mg-EPS/g-VSS. However, in contrast with UASBO-S, the

404

increasing magnitude of both LB-EPS and TB-EPS in the UASBT-S (the right bars of

405

side-by-side bar charts) performed far less with the increase in OLR (see Fig.5 (a) and

406

(b)). It is known that the adverse external conditions could stimulate the

407

microorganisms in the bioflocculation (sludge) to secrete more EPS, which was

408

captured and utilized as substrate by cells for their survival (Wang et al., 2018).

409

Hence, the increased OLR is suggested to be more unfavorable for the 22

410

microorganisms in the UASBO-S. Liao et al. (2001) indicate that the EPS composition

411

could also greatly affect the surface properties of the sludge. As same as the cell

412

surface charge of microorganisms, the amino acids are negatively charged, thus the

413

PN components distributed in the bound EPS are generally negatively charged

414

(Dignac et al., 1998; Laspidou and Rittmann, 2002). On one hand, in the bound EPS,

415

the PN-related substance plays a key role in maintaining the stability of sludge since it

416

is bridged to the metal cation (e.g., Mg2+, Ca2+, Fe3+ , etc.) by an electrostatic bond,

417

which constructed the basic framework of granules. On the other hand, the charge

418

repulsive potential may alienate the EPS flocs and cells, and this effect may be

419

intensified when the PN components are massively accumulated in the bound EPS,

420

further resulting in the fragmentation and floatation of granular sludge. Previous

421

reports indicated that the higher PN/PS ratio (i.e.>1.2) in the bound EPS would

422

increase the potential of sludge flotation and disintegration in the bioreactor (Lu et al.,

423

2018; B. Wang et al., 2017). As shown in Fig.5 (a) and (b), the PN/PS ratio of both

424

LB-EPS and TB-EPS in the UASBT-S falled within the range of 0.5-0.7 and 0.6-0.9.

425

Basuvaraj et al. (2015) indicated that the PN/PS ratio in the bound EPS in a healthy

426

and well granulated sludge was lower than 1.0. This is also consistent with the

427

satisfactory properties of the granules in the the UASBT-S, as mentioned earlier.

428

As shown in Fig.5 (a), in the UASBO-S,the PN/PS ratio in the LB-EPS showed an 23

429

up-ward trend, appeared to fluctuate at 0.6-1.3 at an OLR range of 1-4g-COD/L/d. It

430

is worth noting that the PN/PS ratio drastically dropped to 0.5 when the OLR was

431

enhanced to 8g-COD/L/d. The sharply increased LB-EPS concentration was mainly

432

contributed by the increase in PN, which increased from 7.2 to 24.7mg-EPS/g-VSS.

433

During this period, as mentioned in the section 3.3, quite a number of granules with

434

the paste-like membrane structure were observed in the UASBO-S. It is suggested that

435

the starch and the polysaccharide derivatives adhered on the surface of the granular

436

sludge, embed into LB-EPS under the higher OLR. This explained the decrease in

437

PN/PS ratio of the LB-EPS at the OLR of 8g-COD/L/d. The hydrolyzing bacteria and

438

acidogens adhered on the granular sludge convert the PS component into short-chain

439

carboxylic acids, provide a sufficient carbon source for the acetotrophic archaea

440

distributed in the granular sludge, and further promote the formation of proteins by

441

cells. This may help to clarify the formation mechanism of the paste-like membrane

442

structure on the granules.

443

In the UASBO-S, the PN/PS ratio in the TB-EPS increased from 1.0 to 3.1, with the

444

enhancement of the OLR from 1 to 8g-COD/L/d, as shown in Fig.5 (b). The over-

445

generated EPS covered the surface of granules and clogged the tiny pores on the

446

granules, which may largely limit the substance export (Zheng and Yu, 2007). Thus, it

447

could be hypothesized that the carbohydrate substance adsorbed on the granules was 24

448

converted to acidic metabolites, then transferred and accumulated into the interior of

449

the granules, further exacerbated cell death. The cytoplasm eluted from the dead cells

450

would sharply increase the fraction of negatively charged PN content in the TB-EPS.

451

The charge repulsion force in the microbial flocs would become greater than the van

452

der Waals force, aggravating the sludge disintegration in UASBO-S (Liu et al., 2010).

453

Nielsen and Jahn. (1999) suggested that all the polymers outside the cell wall and free

454

from the murein layer could be classified as bound EPS. In this work, the paste-like

455

membrane structure observed in the UASBO-S is suggested to be the LB-EPS adhered

456

the PS component derived from cell-adsorption. There is no doubt that the setting of

457

pre-acidification unit in the Two-stage process could effectively reduce the EPS

458

generation in the granular sludge in the UASBT-S, helping to overcome the difficult

459

challenges, e.g. granules floatation, disintegration, and washout in the UASB

460

treatment of SW (Lu et al., 2015). Hence, the T-S process with pre-acidification unit

461

is respected to perform application potential in the UASB treatment of the high-

462

strength SW.

463 464

465

3.5 3D-EEM fluorescence spectra of extracted EPS

Since the bound EPS is a blend of various organic polymers with an extremely

25

466

complex composition, it is difficult to perform quantitative analysis for bound EPS

467

using routine test methods. 3D-EEM fluorescence spectroscopy is a sensitive and

468

selective method in the qualitative analysis of the fluorescent substances in a mixture,

469

and the fluorescence intensity is positively correlated with the substance concentration

470

at a specific range. This method is widely employed for the characterization of

471

fluorescent macromolecular content in the EPS (Sheng et al., 2010). Fig.6 exhibits the

472

3D-EEM fluorescence spectra of LB-EPS and TB-EPS extracted from the granular

473

sludge sampled at the "Top" and "Bottom" of the UASBO-S and UASBT-S at an OLR

474

of 4g-COD/L/d. At each 3D-EEM spectra four clear peaks emerged, representing four

475

LB-EPS substances: peak A, Ex/Em=220-230/330-340nm (aromatic protein-like);

476

peak B, Ex/Em=270-290/330-340nm (tryptophan protein-like); peak C, Ex/Em=270-

477

280/450-460nm (fulvic acids); peak D, Ex/Em=350-370nm/440-470nm (humic acids

478

I) (Luo et al., 2014). The fluorescent intensities of aromatic protein-like, tryptophan

479

protein-like, fulvic and humic acids in LB-EPS sampled from "Top" of UASBO-S is

480

higher than in the "Bottom". According to Wang et al. (2016), the humic acids in the

481

bound EPS is positively correlated with the degree of sludge constituents release. This

482

indicates that greater deposition of cellular products and cytoplasmic dissolution

483

occurred thanks to the disintegration of the granular sludge in the UASBO-S. The

484

fluorescent intensities of all the components in the LB-EPS extracted from the 26

485

granules in UASBT-S are significantly lower than those in the UASBO-S. This was in

486

good agreement with the results of LB-EPS content in the two processes.

487

Three peaks were detected in the TB-EPS: peak B; peak E, Ex/Em=320-330/380-

488

390nm (humic acids Ⅱ); and peak F, Ex/Em=420-430/470-480nm (humic acids Ⅲ)

489

(Luo et al., 2014; Wang et al., 2016). It was demonstrated that the much weak

490

fluorescent intensity of humic acids in the TB-EPS in the UASBT-S, was much lower

491

than those in the UASBO-S. All these well verify the earlier hypothesis that 1)

492

accelerated granules disintegration and microbial apoptosis were aggravated in the

493

UASBO-S; 2) The stronger intensity of humic acids in the TB-EPS may source from

494

the cytoplasm elution and conversion of the dead cells in the interior part of granules;

495

3) Pre-acidification effectively relieved the stress response of microorganisms

496

presented in the granular sludge to the adverse environment changes (the enhanced

497

OLR), which was attributed to reduce the massive cell lysis caused by the EPS release

498

and further improve the strength of granular sludge in the UASBT-S.

499 500 501 502

4. Conclusions Comparing with the One-stage UASB reactor, the Two-stage process exhibited both optimal processing performance and stability in the treatment of starch

27

503

wastewater at an OLR range of 1-8g-COD/L/d. The pre-acidification greatly

504

improved the physicochemical properties stability of the granular sludge in terms of

505

granules strength, settling capability, morphological features and PN/PS ratio in the

506

bound EPS. The fatal problems of sludge floatation and disintegration in the UASB

507

reactor was well resolved in the pre-acidification unit equipped Two-stage process,

508

which is recommended in the practical applications of starch wastewater treatment.

509 510

511

ACKNOWLEDGMENTS

This study was financially supported by the Japan Society for the Promotion of

512

Science (JSPS, No. 17J00995). The first author would like to extend special thanks to

513

Mr. Shunsuke Kayamori from the technical division of Tohoku University for his

514

kind technical support.

515 516

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44. Yu, J., Zhao, Ye, Liu, B., Zhao, Yubin, Wu, J., Yuan, X., Zhu, W., Cui, Z., 2016. Accelerated

648

acidification by inoculation with a microbial consortia in a complex open environment.

649

Bioresour. Technol. https://doi.org/10.1016/j.biortech.2016.05.093

650

45. Zhang, F., Yang, H., Guo, D., Zhang, S., Chen, H., Shao, J., 2019. Effects of biomass pyrolysis

651

derived wood vinegar (WVG) on extracellular polymeric substances and performances of

652

activated sludge. Bioresour. Technol. https://doi.org/10.1016/j.biortech.2018.11.064

653

46. Zhang, W., Zhang, F., Li, Y.X., Jianxiong Zeng, R., 2019. Inhibitory effects of free propionic and

654

butyric acids on the activities of hydrogenotrophic methanogens in mesophilic mixed culture

655

fermentation. Bioresour. Technol. https://doi.org/10.1016/j.biortech.2018.10.076

656

47. Zhang, Y., Ma, H., Niu, Q., Chen, R., Hojo, T., Li, Y.Y., 2016. Effects of substrate shock on

32

657

extracellular polymeric substance (EPS) excretion and characteristics of attached biofilm

658

anammox granules. RSC Adv. https://doi.org/10.1039/C6RA20097D

659

48. Zhang, Z.Z., Cheng, Y.F., Xu, L.Z.J., Bai, Y.H., Jin, R.C., 2018. Anammox granules show strong

660

resistance to engineered silver nanoparticles during long-term exposure. Bioresour. Technol.

661

https://doi.org/10.1016/j.biortech.2018.03.024

662

49. Zheng, Y.M., Yu, H.Q., 2007. Determination of the pore size distribution and porosity of aerobic

663

granules using size-exclusion chromatography. Water Res.

664

https://doi.org/10.1016/j.watres.2006.09.015

665

33

Gas meter Biogas Effluent M: Stirring motor P: Peristaltic pump H: Water jacket heater

Gas meter Influent

H

P

Biogas

UASBO-S (6L)

Effluent

One-stage (O-S) process (6L)

M

Influent

Substrate tank (200L) with a condenser 666 667 668

P

Gas meter · M

H

P

H

Acidification unit (6L)

UASBT-S (6L)

Two-stage (T-S) process (12L) Fig.1 Schematic diagram of the Two-stage (T-S) and One-stage (O-S) processes and associated accessories employed in this study.

34

Two-stage (T-S) process

One-stage (O-S) process

MLVSS of granular sludge (g/L)

(a)

20 0 9 8 7

(c)

6 Influent Effluent

5 4 100 80

(e)

60 40 20 0

500

Acetate i-Butyrate i-Valerate

400

Propanate n-Butyrate n-Valerate

(g)

300 200 100 0 80

Not De te cte d

Not De te cte d

60 40 20

(i)

SCOD removal efficiency (%)

40

Influent/Effluent pH

60

Carbohydrate removal efficiency (%)

80

0

670 671 672 673 674 675

80 60 40 CSTR (T-S) Overall

20 0 9

(b)

8 7

(d)

6 5 4

Influent CSTR (T-S) UASB (T-S)

100 80

(f)

60 40 CSTR (T-S) Overall

20 0

500 400

CS TRT-S

Acetate i-Butyrate i-Valerate

Propanate n-Butyrate n-Valerate

(h)

300 200 100 0 80

UAS BT-S

60 40 20

(j)

0

1

669

100

Not De te cte d

VFAs in effluent (mg-AcOH/L)

Carbohydrate removal efficiency (%)

Influent/Effluent pH

SCOD removal efficiency (%)

100

VFAs in effluent (mg-AcOH/L)

VI (3h)

MLVSS of granular sludge (g/L)

Phase (HRT) II (24h) III (12h) IV (6h)

Phase (HRT) III (24h) IV (12h) VI (6h) VII (3h)

2

4

8

1

2

4

8

OLR (g-COD/L/d)

OLR (g-COD/L/d)

Fig.2 The average value of long-term treatment performance in ascending OLR settings, where soluble COD removal efficiencies performed in (a) O-S process, (b) T-S process; Influent and effluent pH measured in (c) O-S process, (d) T-S process; Soluble carbohydrates removal efficiencies performed in (e) O-S process, (f) T-S process; VFA contents measured in (g) O-S process, (h) T-S process; Granular sludge concentration (represented by MLVSS) tested in (i) O-S process, (j) T-S process.

35

UASB reactor in T-S process

UASB reactor in O-S process IV (6h)

V (4h)

VI (3h)

Distribution (%)

80 60

Top R2 ＝0.9985 XC ＝1.29 A＝80.30

R2 ＝0.9995 XC ＝1.34 A＝68.42

Unable to fit

Unable to fit

40 20

Distribution (%)

0 100 80 60

Middle R2 ＝0.9987 XC ＝1.54 A＝58.66

R2 ＝0.9840 XC ＝1.52 A＝68.42

40 20

Distribution (%)

0 100 80 60

Bottom R2 ＝0.9068 XC ＝1.71 A＝57.94

678

R2 ＝0.8531 XC ＝1.91 A＝25.76

Unable to fit

20 1 2 3 4 Diameter (mm)

1

677

R2 ＝0.9999 XC ＝1.69 A＝53.58

40

0

676

Unable to fit

1 2 3 4 Diameter (mm)

2

1 2 3 4 Diameter (mm)

1 2 3 4 Diameter (mm)

4 OLR (g-COD/L/d)

6

I (Start-up)

IV (12h)

V (8h)

VI (6h)

VII (3h)

100

Distribution (%)

III (12h)

80 60

Top R2 ＝0.9985 XC ＝1.29 A＝80.30

R2 ＝0.9992 XC ＝1.36 A＝67.53

R2 ＝0.9998 XC ＝1.33 A＝66.60

R2 ＝0.9987 XC ＝1.54 A＝58.66

R2 ＝0.9417 XC ＝1.53 A＝50.15

R2 ＝0.8568 XC ＝1.64 A＝53.50

R2 ＝0.9068 XC ＝1.71 A＝57.94

R2 ＝0.8941 XC ＝1.75 A＝52.24

R2 ＝0.9932 XC ＝1.74 A＝60.18

1 2 3 4 Diameter (mm)

1 2 3 4 Diameter (mm)

R2 ＝0.9999 XC ＝1.42 A＝64.94

R2 ＝0.9999 XC ＝1.39 A＝64.02

40 20 0 100

Distribution (%)

I (Start-up)

100

80 60

Middle R2 ＝0.9116 XC ＝1.59 A＝56.40

R2 ＝0.9907 XC ＝1.71 A＝50.72

40 20 0 100

Distribution (%)

Phase (HRT)

80 60

1 2 3 4 Diameter (mm)

1 2 3 4 Diameter (mm)

20

0.5

8

R2 ＝0.9920 XC ＝1.81 A＝56.54

40

0

1 2 3 4 Diameter (mm)

Bottom R2 ＝0.9778 XC ＝1.77 A＝53.24

2

1 2 3 4 Diameter (mm)

3 OLR (g-COD/L/d)

4

8

Fig.3 Diameter size distribution of granular sludge taken from the "Top", "Middle" and "Bottom" part of UASBO-S and UASBT-S reactor at each experimental period. Column chart represents the distribution ratio of different particle size: blue is 0.35-0.5mm; yellow is 0.5-1.0mm; grey is 1.02.0mm; orange is 2.0-2.8mm and green is 2.8-4.0mm. Green curves were fitted according to the Gaussian distribution model.

36

(e)

Cemented granules

UASBO-S Paste-like structure

Granules floatation

(b)

5mm

(d)

1mm

679

5mm

0.97 8.55

100 80

43.12

60 6.64

40

Granules with a smooth surface 1mm

0

UASB UASB (SP) O-S 1.49 8.69

100 80

40.60

60 6.66

40

42.55

20 0

UASB UASB (TP) T-S

Fig.4 Morphological observations and elemental compositions analysis of the granular sludge at an OLR of 4g-COD/L/d. 37

C H O N S

40.72

20

(h)

(f)

UASBT-S

Well-settling granules

(g) Elemental percent composition (%)

(c)

Elemental percent composition (%)

(a)

C H O N S

30

(a) 4 0

681 682

EPS-PS PN/PS (T-S)

1.5

3 0

1.0 1 .0

20 10 0 300 250

UASBO-S UASBT-S 2 0

0.5

1 0

0.0 4.0

0

0 .0

3 0

(b) 2 50

4 .0

EPS-PN PN/PS (O-S)

EPS-PS PN/PS (T-S)

3.0 3 .0

200 150 100

2 0

UASBO-S UASBT-S

2.0

1 50

2 .0

1 0

1.0 1 .0

50 0

5 0

0.0 0 .0

0

Start-up 680

2 .0

EPS-PN PN/PS (O-S)

PN/PS ratio

40

2.0

5 0

PN/PS ratio

LB-EPS (mg-EPS/g-VSS) TB-EPS (mg-EPS/g-VSS)

50

1

2 4 OLR (g-COD/L/d)

8

Fig.5 Variation of EPS contents and PN/PS ratio in the granular sludge sampled from the "Bottom" of UASBO-S and UASBT-S: (a) loosely bound EPS (LB-EPS); (b) tightly bound EPS (TB-EPS)

38

a

440 420

b

UASBO-S @OLR=4g-COD/L/d 0

Top

Peak D

400

420 400

4

380

Ex (nm)

12

Peak B

320

Peak C

16

300 280

LB-EPS

240

400

24

350

400

450

500

550

420

4

400

16

300

Ex (nm)

Ex (nm)

12

320

300

350

400

450

500

550

600

28

UASBO-S @OLR=4g-COD/L/d Em (nm)

Top

d

0

550

600

Em (nm)

28

0 4 8

340

12

320

16

300

20

220 250 440

24

LB-EPS 300

350

400

450

500

550

600

28

UASBT-S @OLR=4g-COD/L/d

Em (nm)

0

Top

32

320 300

Peak E

260

300

350

Bottom

400

500

550

600

Em (nm)

32

320 300

48

260

TB-EPS 450

340

280

48

Peak B

240

16

380

Ex (nm)

Ex (nm)

500

360

340

280

TB-EPS

240 220 250 440

64 0

420

400

300

350

Bottom

400

450

500

550

600

Em (nm)

64

0

400

380

380

16

360

16

360

340

32

320 300 280

Ex (nm)

Ex (nm)

450

360

420

16

Peak F

360

48

260

TB-EPS

240 300

350

400

450

500

550

600

340

32

320 300 280

48

260

TB-EPS

240

64

Em (nm)

685

Bottom

400

24

400

380

684

350

240

400

683

300

260

24

LB-EPS

240

220 250

LB-EPS

280

20

260

420

20

380

340

220 250 440

16

300

0

8

420

12

320

440

360

440

8

340

220 250

600

Em (nm)

Bottom

360

240

28 300

280

c

4

260

380

220 250

0

Top

280

20

Peak A

260

Ex (nm)

8

340

420

UASBT-S @OLR=4g-COD/L/d

380

360

220 250 440

440

220 250

300

350

400

450

500

550

600

64

Em (nm)

Fig.6 3D-EEM fluorescence spectra of the TB-EPS and LB-EPS, extracted from granular sludge sampled from the "Top" and "Bottom" of the UASBO-S and UASBT-S at an OLR of 4g-COD/L/d.

39

686 687

Table 1. Long-term operational conditions of the O-S and T-S process in this study. Phase

I (Start-up)

II

III

IV

Duration time (days)

0-40

41-80

81-120

121-160

Temperature (°C ) Influent flow rate (L/h)

35±1 0.25

35±1 0.25

35±1 0.5

35±1 1

One-stage

24

24

12

6

Two-stage

48

48

24

12

One-stage

1

1

2

4

Two-stage

0.5

0.5

1

2

HRT (h) OLR (g-COD/L/d) Phase

35 1

I

II

III

IV

V

Soluble COD (mg/L)

898±99

895±139

814±59

854±59

917±55

Soluble PS (mg/L)

821±179

957±167

886±101

839±38

829±51

SS (mg/L)

335±35

344±36

324±28

374±9

373±15

NaHCO3 dosage (mg/L)

1000

1000

1250

1500

1750

pH (on average)

7.58

7.59

7.75

7.98

7.61

688 689 690 691 692 693 694 695 696 697 698 699 700

161

Table 2. Phased quality parameters of the synthetic SW employed in this study.

701 702

40

703

Graphic abstract Electrostatic repulsion

biogas bubble

Cell Dispersive part Protein Polysaccharide T-B EPS Granular sludge L-B EPS

Fluctuations in bound EPS

One-stage UASB process

Satisfactory treatment performance

Starch wastewater

M

Pre-acidification

Granular sludge

Two-stage process

704 705 706

Highlights

707

• Two UASB processes were compared in treatment of low-strength starch wastewater.

708

• Floatation and disintegration of granular sludge occurred in the One-stage UASB.

709

• Gelatinous attachment to the granules exerted harmful effects on process stability.

710

• The pre-acidification was beneficial for the stabilization of the granular sludge.

711 712 713

The authors declare that there are no conflicts of interest in this work.

41

S

714 715

Credit Author Statement for BITE-D-19-04413R1

716 717

Jiang Wu: Resources, Conceptualization, Methodology, Investigation, Software, Writing - Original

718

Draft.

719

Bo Jiang: Investigation, Data Curation.

720

Bo Feng: Investigation.

721

Lu Li: Software, Data Curation.

722

Siti Nur Fatihah Moideen: Data Curation.

723

Hong Chen: Investigation

724

Chaimaa Mribet: Writing: Review & Editing.

725

Yu-You Li: Supervision, Conceptualization, Writing - Review & Editing

726

42