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Anaerobic treatment of glutamate-rich wastewater in a continuous UASB reactor:

Effect of hydraulic retention time and methanogenic degradation pathway

Hong Chen, Yanxiao Wei, Chenglei Xie, Hong Wang, Sheng Chang, Ying Xiong,
Chunyan Du, Benyi Xiao, Guanlong Yu

PII: S0045-6535(19)32912-1
DOI: https://doi.org/10.1016/j.chemosphere.2019.125672
Reference: CHEM 125672

To appear in: ECSN

Received Date: 25 September 2019

Revised Date: 27 November 2019
Accepted Date: 14 December 2019

Please cite this article as: Chen, H., Wei, Y., Xie, C., Wang, H., Chang, S., Xiong, Y., Du, C., Xiao,
B., Yu, G., Anaerobic treatment of glutamate-rich wastewater in a continuous UASB reactor: Effect of
hydraulic retention time and methanogenic degradation pathway, Chemosphere (2020), doi: https://
doi.org/10.1016/j.chemosphere.2019.125672.

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Anaerobic treatment of glutamate-rich wastewater in a continuous UASB

reactor: Effect of hydraulic retention time and methanogenic degradation

pathway

Hong Chen a,b,c, Yanxiao Wei a, Chenglei Xie c, Hong Wang a, Sheng Chang d, Ying

Xiong a, Chunyan Du a, Benyi Xiao b,*, Guanlong Yu a

a.
Key Laboratory of Dongting Lake Aquatic Eco-Environmental Control and

Restoration of Hunan Province, School of Hydraulic Engineering, Changsha

University of Science & Technology, Changsha 410004, China;

b
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,

Beijing 100085, China

c
Department of Civil and Environmental Engineering, Graduate School of

Engineering, Tohoku University, Sendai 980-8579, Japan

d
School of Engineering, University of Guelph, Guelph N1G 2W1, Ontario, Canada

* Corresponding author.

E-mail: byxiao@rcees.ac.cn
 Euryarchaeota
(S)-Glutamate acid

Archaea
Methanosaeta
Methanobacterium Clostridium pascui
Methanosarcina
Anaeroarcus burkinensis
others (s)-Citramalate Acidaminococcus
Firmicutes sp.
Clostridium
Anaeromusa Propionate
Syntrophomonas
Pyruvate
Acetoanaerobium
Acidaminococcus
Bacteroidetes Syntrophobacter sp.

Bacteria
Lentimicrobium Butyrate
DQ677001_g
Proteobacteria Syntrophomonas sp. Acetoanaerobium H2/CO2
Syntrophobacter
Firmicutes Klebsiella sp.
Synergistetes
Proteobacteria
Synergistetes Acetate
Bacteroidetes AF280863_g Methanobacterium beijingense
Saccharibacteria TM7 Saccharibacteria_TM7 Methanosaeta concilii Methanobacterium
others Saccharimonas
others subterraneum
Glutamate
80 70 60 50 40 30 20 10 0 CH4 Methanobacterium formicicum
feeding Percentage (%)
UASB reactor Microbial Communities (Phase Ⅶ) Proposed methanogenic degradation pathways
 1 Anaerobic treatment of glutamate-rich wastewater in a continuous UASB reactor:

2 Effect of hydraulic retention time and methanogenic degradation pathway

4 Abstract

5 To investigate the anaerobic treatment efficiency and degradation pathways of

6 glutamate-rich wastewater under various hydraulic retention times (HRTs), a lab-scale

7 upflow anaerobic sludge blanket (UASB) reactor was operated continuously for 180

8 days. Results showed that high chemical oxygen demand (COD) removal efficiencies

9 of 95.5%–96.5% were achieved at HRTs of 4.5 h to 6 h with a maximum methane

10 yield of 0.31 L-CH4/g-COD. When the HRT was shortened to less than 3 h, the

11 removal performance of the reactor declined. There also was an excessive

12 accumulation of volatile fatty acids, which implies that an appropriately small HRT is

13 applicable to the UASB reactor treating glutamate-rich wastewater. Methanogenic

14 degradation of glutamate in the UASB reactor depended on the HRT applied, and the

15 typical methane-producing capability of the sludge at an HRT of 3 h, in descending

16 order, was acetate > glutamate > butyrate > H2/CO2 > valerate > propionate.

17 Clostridium and Methanosaeta were predominant in the glutamate-degrading sludge.

18 At least three degradation pathways most likely existed in the UASB reactor, and the

19 pathway via 3-methlaspartate by Clostridium pascui was expected to be dominant.

20

21 Keywords: 3-methylaspartate pathway; Biodegradation; Glutamate fermentation;

22 Methanogenesis; Upflow anaerobic sludge blanket

1
23 1. Introduction

24 Since it was first commercially produced as a flavor-enhancing additive in Japan in 1909,

25 monosodium glutamate (MSG) has been widely used in the food industry (Ault, 2004).

26 As the largest MSG producing country worldwide, China produces 2.2 million tons of

27 MSG per year, accounting for approximately 80% of the total global production (Dong et

28 al., 2018). Meanwhile, a large amount of wastewater is produced during MSG production,

29 with high concentrations of chemical oxygen demand (COD) (10,000–40,000 mg/L),

30 NH4+-N (15,000–25,000 mg/L), and sulfate (15,000–30,000 mg/L), and a very low pH

31 (approximately 2.0) (Xue et al., 2008). Sulfate reduction by sulfate reducing bacteria that

32 competes with methane producing archaea occur with the exist of sulfate, which

33 influences on the bioenergy recovery efficiency and even the overall performance by the

34 produced hydrogen sulfide (Lu et al., 2016). To avoid serious environmental pollution by

35 MSG wastewater, the development of cost-effective and environmental-friendly treatment

36 technologies has attracted researchers’ interest (Jiang et al., 2015; Singh et al., 2009;

37 Tseng & Lin, 1990; Yao et al., 2010). Of these studies, anaerobic treatment is considered

38 among the most promising technologies, given its superiority in energy recovery, less

39 secondary pollution, and its wide application (Fang & Zhang, 2015; Feng et al., 2018;

40 Han et al., 2017).

41

42 Since it was developed in the 1970s, upflow anaerobic sludge blanket (UASB) reactors

43 have been widely utilized for the treatment of various types of wastewater, particularly

44 high strength food processing and beverage wastewater (Li et al., 2015; Chen et al.,

45 2019a; Olivares et al., 2016). Several studies have investigated the feasibility of using a

46 UASB reactor for the treatment of MSG wastewater; these studies examined the startup

2
47 performance of the reactor, as well as its biological degradation activity and kinetic

48 parameters (Cao et al., 1992). Tseng & Lin (1990) obtained a maximal COD removal

49 efficiency of 65% for treating the MSG wastewater using an anaerobic biological

50 fluidized bed reactor. Nevertheless, in these studies, the washout of granules was

51 observed, and the obtained kinetic constants could not be used to describe the UASB

52 process (Cao et al., 1992; Tseng & Lin, 1990). In practice, hydraulic retention time (HRT)

53 is one of the most important design and operational parameters of UASB reactors (Chen

54 et al., 2018a; Kim et al., 2014). To scale down wastewater treatment projects, a relatively

55 small HRT is commonly proposed, which corresponds with a high upflow velocity for the

56 UASB reactor. However, few studies have investigated the effect of HRT for the

57 long-term anaerobic treatment of MSG wastewater in UASB reactors. It remains

58 necessary to evaluate the effect of HRT on MSG wastewater treatment in a UASB reactor

59 for continuous long-term operation.

60

61 On the other hand, to better understand the inherent removal mechanisms of anaerobic

62 wastewater treatment, many studies have focused on changes in the microbial community

63 as well as pollutant degradation pathways under anaerobic conditions (Chen et al., 2019b;

64 Lu et al., 2017; Sudmalis et al., 2018; Tian et al., 2015). Investigations on microbial

65 responses and metabolic pathways can reveal the process mechanism of the bioreactor

66 system such as changes in the microbial community structure in long-term competition

67 (Wu et al., 2018) and recovery mechanisms of biogas production under ammonia

68 inhibitions (Chen et al., 2018b). Generally, anaerobic degradation of amino-containing

69 pollutants occurs via Stickland fermentation (Batstone et al., 2003; Fang & Zhang, 2015).

70 For glutamate fermentation, at least five different pathways have been verified thus far,

3
71 including two coenzyme B12-dependent 3-methylaspartate pathways, a

72 2-hydroxyglutarate pathway, and pathways via 4-aminobutyrate – radical formation by

73 one-electron oxidation and via 5-aminovalerate – transient two-electron oxidation of

74 5-hydroxyvaleryl-CoA (Buckel, 2001; Buckel & Thauer, 2013). Bacterial orders,

75 including Clostridiales and Fusobacteriales, were identified in soil, sewage sludge, both

76 marine and freshwater sediments, and in the gastrointestinal tract of animals. Some

77 closely related anaerobic bacteria (Clostridium tetani, Clostridium tetanomorphum, and

78 Clostridium pascui) are involved in the fermentation of glutamate to acetate, butyrate,

79 carbon dioxide, and ammonia (Buckel, 2001), but those species have never been reported

80 in a UASB reactor for glutamate degradation. Hence, it is necessary to investigate

81 microbial community changes and degradation pathways to understand the process

82 mechanisms of MSG wastewater treatment by UASB reactors.

83

84 The aim of this study was to discover the effect of one of the key operational parameters,

85 HRT, on anaerobic treatment performance and removal mechanisms of glutamate-rich

86 wastewater. A UASB reactor operated continuously for 180 days under various HRTs

87 ranging from 24 h to 2 h. The changes in microbial community structures between startup

88 and later operational periods were characterized by 16S rDNA gene sequencing. within

89 addition to specific methanogenic activity (SMA) tests of granular sludge with different

90 substrates, methanogenic pathways for degrading glutamate in glutamate-rich wastewater

91 were also explored.

92

93 2. Materials and Methods

94 2.1 Experimental apparatus

4
 95 A schematic diagram of the experimental set-up is illustrated in Fig. 1. A lab-scale UASB

96 was utilized and consisted of a substrate tank, peristaltic pump, water bath system, UASB

97 main body, gas buffer bottle, and wet gas flow meter. The UASB main body consisted of

98 a gas-liquid-solid separator and a cylindrical reaction zone, which was enclosed by a

99 plexiglass cylinder with an internal diameter of 100 mm, a reaction zone height of 800

100 mm, and an effective working volume of 6 L.

101 Fig. 1.

102

103 2.2 Inoculum and synthetic wastewater

104 The reactor was inoculated with 2 L of fresh sludge from the anaerobic unit of a

105 wastewater treatment plant in Changsha City, China, and 2 L of granular sludge from a

106 UASB reactor treating practical starch wastewater in Inner Mongolia, China. The

107 granular sludge had a mixed liquor suspended solids concentration of 80 g/L and a mixed

108 liquor volatile suspended solids concentration of 48 g/L (Supplementary data). Synthetic

109 wastewater was prepared by analytical (S)-glutamate and the following minerals: COD

110 2000 mg/L, K2HPO4 250 mg/L, KH2PO4 100 mg/L, KCl 300 mg/L, MgCl·6H2O 50 mg/L,

111 CoCl2·6H2O 0.4 mg/L, CaCl2 15 mg/L, FeCl2·4H2O 3.56 mg/L, (NH4)6Mo7O24·4H2O

112 0.65 mg/L, NiCl2·6H2O 0.81 mg/L, ZnC12 0.60 mg/L, and CuC12·2H2O 0.3 mg/L.

113

114 2.3 Experimental procedure

115 The operational conditions of the reactor are shown in Table 1. The UASB reactor

116 operated continuously for 180 days under various HRTs that were categorized into seven

117 phases (I–VII). These phases corresponded with the HRTs of 48 h, 24 h, 12 h, 6 h, 4.5 h,

118 3 h, and 2 h, respectively. The influent COD concentration was set at 2000 mg/L. Both

5
119 the operational temperature and the influent pH value remained constant throughout the

120 different HRT phases.

121

122 Table 1

123

124 2.4 Analytical methods

125 The total COD, NH4+-N, and alkalinity in the influent and the effluent and total

126 suspended solid (TSS), volatile suspended solid (VSS), and extracellular polymeric

127 substances (EPS) of anaerobic granules were determined according to references (Lu et

128 al., 2015; Xiao et al., 2018). The pH value was measured with a pH meter (PHSJ-3F,

129 Shanghai). The daily biogas production was measured with a laboratory biogas wet gas

130 flow meter (JH-LMF-1, Jinzhiye). Biogas composition and volatile fatty acids (VFAs)

131 were analyzed using gas chromatography with a flame ionization detector and a thermal

132 conductivity detector (GC9790II, Fuli). Proteins (PN) and polysaccharides (PS) in the

133 granules were measured with the Lowry method and the phenol/H2SO4 method,

134 respectively (Lu et al., 2015; Wu et al., 2018). The particle size of the granular sludge

135 was evaluated with a standardized sieve series, while the mean settling velocity was

136 evaluated with settling column tests (Lu et al., 2017). Both the morphology and the

137 microstructure of the granules were analyzed with scanning electron microscopy

138 (S-3000N, Hitachi).

139

140 2.5 Specific methanogenic activity tests

141 Granular sludge was taken from the UASB reactor for the specific methanogenic activity

142 (SMA) test; 120 mL serum bottles were also required and contained methanol, acetate,

6
143 propionate, n-butyrate, n-valerate, (S)-glutamate, and H2/CO2 as a substrate, respectively.

144 The SMA analysis involved filling each serum bottle with 10 mL of granular sludge and

145 50 mL of anaerobic sludge (inoculum), adding the various substrates, and adjusting the

146 pH values of each to 7. Each serum bottle was topped off with the trace element nutrient

147 solution to make a total solution volume of 80 mL, and then the serum bottles were sealed

148 with rubber stoppers and secured by aluminum crimps. The nutrient solution was boiled

149 for 2 h prior to use to remove any dissolved oxygen present and then cooled to room

150 temperature under a nitrogen atmosphere. The initial concentration for the serum bottles

151 was maintained at 2000 mg-COD/L. Oxygen in the headspace of the bottles was purged

152 with nitrogen gas for 5 min. However, the bottle containing the additional H2/CO2

153 substrate required H2/CO2 (80:20, v/v) gas to remove oxygen in the headspace, and an

154 ultimate gas pressure of 1.4 atm was maintained by injecting an additional 30 mL of

155 H2/CO2. Then, 0.25 mL of 2000 mg/L Na2S·9H2O solution was injected into each bottle

156 to maintain an absolute anaerobic condition. Finally, all serum bottles were placed in an

157 incubator (100±5 rpm, 35±1 °C). After waiting 5 min for the temperature of each bottle to

158 increase to the set value, the headspace was vented using a syringe to release the pressure

159 caused by the thermal expansion. Biogas production and composition were measured for

160 standard conditions at intervals of 2 h to 6 h. Each experiment was replicated to ensure

161 reliability.

162

163 2.6 Microbial community analyses

164 Biomass samples from the reactor were collected during phases I (Day 35) and VII (Day

165 180) to analyze the microbial communities by 16s rRNA high-throughput sequencing.

166

7
167 Microbial DNA was extracted from samples with an E.Z.N.A.® soil DNA Kit (Omega

168 Bio-tek, USA) according to the protocols of the manufacturer. The final DNA

169 concentration and purification were determined with a NanoDrop 2000 UV-vis

170 spectrophotometer (Thermo Scientific, USA). The quality of the DNA was checked using

171 1% agarose gel electrophoresis. The V3-V4 hypervariable regions of the bacterial and the

172 archaeal 16S rRNA gene were individually amplified with the primers (Supplementary

173 data) using a polymerase chain reaction (PCR) thermal cycler system (GeneAmp 9700,

174 ABI, USA). The PCR reactions were conducted in the following manner: 3 min of

175 denaturation at 95 °C, 27 cycles of 30 s at 95 °C, 30 s for annealing at 55 °C, 45 s for

176 elongation at 72 °C, and a final extension at 72 °C for 10 min. The PCR reactions were

177 performed in triplicate with a 20 µL mixture containing 4 µL of 5 × FastPfu Buffer, 2 µL

178 of 2.5 mM dNTPs, 0.8 µL of each primer (5 µM), 0.4 µL of FastPfu polymerase, and 10

179 ng of template DNA. The resulting PCR products were extracted from a 2% agarose gel,

180 further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA),

181 and quantified using the QuantiFluor™-ST (Promega, USA) according to the protocols of

182 the manufacturer.

183

184 Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an

185 Illumina MiSeq platform (Illumina, San Diego, USA) according to standard protocols

186 (Majorbio, Shanghai, China). Raw fastq files were demultiplexed, quality-filtered by

187 Trimmomatic, and merged using FLASH with the following criteria: (i) the reads were

188 truncated at any site receiving an average quality score <20 over a 50 bp sliding window;

189 (ii) primers were exactly matched allowing for two nucleotide mismatches, and reads

190 containing ambiguous bases were removed; (iii) sequences with an overlap longer than

8
191 10 bp were merged according to their overlap sequence.

192

193 Operational taxonomic units (OTU) were clustered with a 97% similarity cutoff using

194 UPARSE (version 7.1, http://drive5.com/uparse/), and chimeric sequences were identified

195 and removed using UCHIME. After generating an OTU table with the phylogenetic

196 information of each OTU and its abundance in each sample, OTU analysis and the alpha

197 diversity calculation were both completed using the free online platform, Majorbio

198 I-Sanger Cloud Platform (www.i-sanger.com). Similarity searches for the obtained

199 sequences were performed via the EzBioCloud server (http://www.ezbiocloud.net) (Yoon

200 et al., 2017). Raw sequencing reads were deposited in the Sequence Read Archive at the

201 NCBI under accession number PRJNA566074.

202

203 2.7 Calculations

204 The methane and the biogas production, in terms of standard temperature and pressure

205 (STP), were used for the calculations of biogas production rate (BPR), methane yield, and

206 methane production rate (MPR).

207

208 A modified Gompertz equation was employed to estimate the maximum hydrogen

209 production rate in Eq. (1).

210 (1)

211 where P is the cumulative methane production, mL-CH4; Rmax is the maximum MPR,

212 mL-CH4/d; Pm is the methane production potential, mL-CH4; e = 2.71828; λ is lag-phase

213 time, d; and t is the time, d.

214

9
215 The maximum SMA was determined as maximum rate of methane production, expressed

216 as grams COD of methane per gram sludge VSS per day in Eq. (2).

217 (2)

218 where Sm is the maximum SMA, g-COD CH4/g VSS/d; C’o is the conversion coefficient

219 of the volume of methane containing saturated steam to COD mass, 394 mL-CH4/g-COD

220 CH4 (25 °C); VR is the volume of the reaction liquid in the SMA tests, mL; and VSS is the

221 sludge concentration, g VSS/L.

222

223 The total VFA (VFAs) was calculated based on the total molar concentration of all the

224 acids tested, and the total VFA mass concentration was calculated using the following

225 equation (Cheng et al., 2018):

226 (3)

227 Here, MAcetic acid is the molecular weight of acetic acid, and C is the molar concentration

228 of the acid, mmol/L.

229

230 3. Results and Discussion

231 3.1 Overall performance under different HRTs

232 The overall performance of the UASB reactor from phases I to VII, in terms of pH,

233 alkalinity, COD, NH4+-N, VFA, biogas production, and methane content, are presented in

234 Fig. 2.

235

236 Fig. 2

237

238 During the start-up stage (phase I), the operational conditions (including the pH, effluent

10
239 NH4+-N, and alkalinity) gradually stabilized after 35 days of operation. The COD

240 removal efficiency increased to greater than 90%, and the methane percentage of the total

241 biogas increased to 70% at the end of phase I. However, the concentrations of the VFAs

242 drastically decreased to less than 200 mg/L in the first 10 days and continued to decline,

243 which indicated that methanogens in the granular sludge gradually grew and actively

244 functioned to decompose the VFAs under the current operational condition. Therefore,

245 the UASB reactor performed satisfactorily during the start-up stage for treating the

246 synthetic MSG wastewater.

247

248 During phases II–VII, the HRT was reduced from 24 h to 2 h, while all the other

249 parameters remained unchanged. As shown in Fig. 2a, the effluent pH stabilized at

250 approximately 7.1 to 7.3, with a slight increase in NH4+-N acclimation during the

251 anaerobic digestion process using glutamate as the sole carbon and nitrogen source. Fig.

252 2b shows that the effluent NH4+-N concentration slowly increased over time, and during

253 phase VII it finally reached a maximum value of 195±15 mg/L, which was still below the

254 minimum inhibitory concentration of ammonia (Chen et al., 2018b). The effluent total

255 alkalinity stabilized at 1107.7–1455.8 mg-CaCO3/L until the HRT was reduced to 4.5 h,

256 while a marked increase in alkalinity was observed at an HRT of 3 h and eventually

257 reached 2055.3–2453.5 mg-CaCO3/L with a bicarbonate alkalinity of 956.8–1426.1 mg/L

258 at an HRT of 2 h (Fig. 2c). The elevated alkalinity in the effluent reflected the dynamic

259 equilibrium between consumption and re-generation of inorganic carbon (HCO3-) and

260 ammonium (NH4+), which was enough to maintain pH stability (Lu et al., 2015).

261

262 A stable COD removal efficiency of approximately 95% was obtained during phases II–V

11
263 (Fig. 2d) but decreased to approximately 78% during phase VI (HRT = 3 h) and

264 approximately 67% in phase VII. In comparison, more than 93% of COD of dimethyl

265 phthalate was removed (Kong et al., 2018, Jia et al., 2007); and the highest COD removal

266 was 76.6% in biodegradation of MSG wastewater, which was lower than the removal

267 efficiency (97.9%) in the previous study (Chen et al., 2020). Moreover, only a small

268 amount of VFAs emerged during phase V (below 200 mg/L) and a relatively greater

269 percentage during phase VI, but a massive amount of VFAs accumulated during phase

270 VII (400–1000 mg/L) (Fig. 2e). During phase V, the COD removal efficiency initially

271 decreased to 78.2%, then gradually increased and stabilized at 95.5%, which

272 corresponded to a decrease in the VFA concentration in the effluent from 179.3 mg/L to

273 52.1 mg/L. Generally, low VFA concentrations (less than 200 mg/L) have minor impact

274 on the performance of bioreactors (Fang & Zhang, 2015). However, the performance of

275 the reactor severely deteriorated during phase VII at an HRT of 2 h when excessive VFAs

276 accumulated (greater than 500 mg/L), thereby depressing the activity of the functional

277 bacteria in the reactor.

278

279 As shown in Fig. 2f, both the methane content and the BPR were substantially affected by

280 the changes in the HRTs. During the entire operational period, the BPR increased with a

281 decrease in the HRT, which was similar to the increase in the organic loading rate while

282 maintaining the unchanged influent COD concentration. However, the methane content in

283 the biogas stabilized at approximately 73% under HRTs from 48 h to 4.5 h, followed by a

284 small decrease at an HRT of 3 h and a drastic decrease to 57% at an HRT of 2 h. Methane

285 production in a UASB or an EGSB at a very short HRT was easily inhibited by the

286 accumulation of VFAs (Fig. 2e), ammonia, and other inhibitors or by other process

12
287 problems. For instance, a large amount of foam produced could block the gas-liquid-solid

288 separator, which could result in the escape of biogas from the outlet and subsequent

289 attenuation of the collection efficiency of the biogas (Lu et al., 2015; Rajagopal et al.,

290 2013).

291

292 From the overall performance of the UASB reactor, we concluded that an operational

293 HRT of less than 3 h is not suitable for treating synthetic MSG wastewater that is

294 equivalent to a maximum organic loading rate of 16 g-COD/L/d, which was similar to the

295 organic loading rates proposed by other researchers for a stable process (Li et al., 2015;

296 Lu et al., 2017).

297

298 3.2 Effect of HRT

299 The effect of different HRTs on the performance of the UASB, in terms of COD removal

300 efficiency, organic removal rate (ORR), NH4+-N and VFA production, methane yield, and

301 MPR, were evaluated when the UASB reactor reached a relatively steady performance

302 during each phase (Fig. 3). An increase in NH4+-N production corresponded with a

303 decrease in the HRT (Fig. 3a). Ammonium generated during the glutamate degradation

304 process formed NH4HCO3 with carbon dioxide, which helped to neutralize the acids and

305 maintain the pH within a stable range (Figs. 2a and 2c) (Fang & Zhang, 2015; Rajagopal

306 et al., 2013). Few VFAs emerged in the reactor during phases II–IV, but an excessive

307 amount of VFAs accumulated during phase VII (Fig. 3b). When the total VFAs reached

308 approximately 600 mg-HAc/L during phase VII, the methanogenesis was easily

309 overwhelmed, and then the glutamate fermentation process was greatly inhibited (Cheng

310 et al., 2018). Under a short operational HRT (< 3 h), the conversion of VFAs became a

13
311 rate-limiting step in the UASB reactor for MSG wastewater treatment.

312

313 Fig. 3

314

315 Throughout the entire operational period, with changing HRTs from 48 h to 2 h, the ORR

316 correspondingly increased from 1.0±0.1 g-COD/L/d to 16.6±2.1 g-COD/L/d (Fig. 3c).

317 The mean COD removal efficiency were 91.7±3.4%, 94.6±5.7%, 94.5±2.8%, 95.0±4.2%,

318 and 96.3±3.4% under the HRTs of 48, 24, 12, 6, and 4.5 h, respectively. However, the

319 mean COD removal efficiency decreased to 78.8% and 67.0% during phases VI and VII,

320 respectively. The HRT had a significant effect on the MPR (it increased from 0.25±0.06

321 L-CH4/L/d to 4.34±0.41 L-CH4/L/d) and the methane yield (first it increased from

322 0.26±0.09 L/g-COD to 0.31±0.02 L/g-COD and then decreased to 0.18±0.03 L/g-COD)

323 (Fig. 3d). The deteriorated methane production under low HRTs indicated that the

324 performance of the UASB system was greatly dependent upon the HRT applied during

325 the MSG wastewater treatment.

326

327 3.3 COD mass balance analyses

328 The COD mass balance was calculated for phases II–VII (Fig. 4), which accounted for

329 the COD flowing in and out of the system and the COD converted into methane, but it

330 neglected the COD that was converted into biomass (Lu et al., 2015). The distribution of

331 COD mass flows varied with HRT. The minimum percentage of COD mass flow through

332 the effluent was 7.5%±3.7% (phase III) with a corresponding mass flow percentage of

333 80.1%±4.6% through the methane production. The maximum percentage of COD mass

334 flow through methane production was 83.3%±3.2% (HRT = 6 h) and 82.7%±9.0% (HRT

14
335 = 4 h), which indicated that the best energy recovery from the MSG wastewater was

336 under these HRT conditions (Kim et al., 2014). However, during phases VI and VII (with

337 HRTs of 3 h and 2 h, respectively), the COD mass flow through the effluent increased to

338 24.5%±4.1% and 34.7%±4.0%, respectively. In terms of energy recovery here, the

339 maximum percentage of COD converted into methane was 83.3%±3.2%, which was

340 similar to that of 81.7% for the treatment of starch wastewater (Lu et al., 2015). From the

341 COD mass balance analysis, it was apparent that operating a UASB reactor at an HRT

342 less than 4.5 h is unfavorable for energy recovery from MSG wastewater.

343

344 Fig. 4

345

346 3.4 Characteristics of granular sludge

347 For a UASB applied to wastewater treatment, the formation of granular microbial

348 aggregates played a crucial role in maintaining a stable process performance (Lu et al.,

349 2017; Sudmalis et al., 2018; Tan et al., 2018). In this study, granular sludge was collected

350 from the UASB reactor during each phase to characterize its properties in terms of VSS,

351 VSS/TSS, particle size distribution, settling velocity, EPS content, PN/PS ratio, and

352 microstructure. The results are shown in Fig. 5 and Fig. S1 in Supplementary data. Under

353 various HRTs from 48 h to 2 h, the VSS of the granular sludge first increased from 34

354 g-VSS/L (phase I), to a maximum value of 46 g-VSS/L (phase V), and then decreased to

355 38 g-VSS/L (phase VII) (Fig. 5a). The VSS/TSS ratio also showed a stepwise increase

356 during the first operational days (phases I–VI) with a slight decrease at the end of the

357 operational period (phase VII). Both the particulate organic matter and the total

358 particulate solids in the UASB reactor were relatively high during phases III–VI, which

15
359 implied that the biomass concentration of the granular sludge in the UASB reactor was

360 very high during these days. The increase in VSS from phase I to phase IV indicated a

361 high bioactivity and reproductive capacity of biomass during these operational phases

362 (Liang et al., 2007).

363

364 Fig. 5

365

366 Both the particle size distribution and the settling velocity of the granular sludge varied

367 with different HRTs (Fig. 5b). The percentage of small particles (≤ 1 mm) increased from

368 63% (phase I) to 72% (phase II), then decreased to a minimum value of 46% (phase IV)

369 before it finally reached 78% (phase VII). The percentages of large particles (≥ 1 mm) at

370 various HRTs from 12 h to 4.5 h (phases III–V) were greater than during the start-up

371 stage (phase I). Fig. 5b shows the average settling velocities of the granular sludge during

372 phases II–V were also faster than during phase I. The superior properties in particle size

373 distribution and settling velocity with the granular sludge obtained during phases III–V

374 aligned with the stable overall performance of the UASB reactor (Fig. 2). Additionally, a

375 large percentage of small particles with very slow settling velocities occurred in the

376 reactor during phases VI–VII, which implied poor granulation or collapse of the granular

377 structure under those HRT conditions. The collapse of the granular structure in a UASB

378 reactor could result in deterioration of the effluent quality due to biomass washout and

379 poor dewatering of the sludge (Kim et al., 2014; Pol et al., 2004). Although a short HRT

380 could help promote granulation in UASB reactors (Lu et al., 2017; Sudmalis et al., 2018),

381 this study demonstrated that extremely short HRTs would be detrimental to the formation

382 of granules in the UASB reactor.

16
383

384 The EPS contents and PN and PS portions of the tightly bound EPS (TB-EPS) extracted

385 from the granular sludge under various HRTs are shown in Fig. 5c. The EPS content

386 increased from 120 mg/L (phase I) to the maximum value of 210 mg/g (phase III),

387 maintained at 205 mg/g (phase IV) and 195 mg/g (phase V), but decreased to 160 mg/g

388 (phase VII). The EPS can help to promote adhesion of microorganisms through chemical

389 bonds or physical entanglement, and granules with a higher TB-EPS content usually

390 possess better mechanical strength and physical stability (Basuvaraj et al., 2015; Pol et al.,

391 2004). In this regard, the granular sludge during phases III–V was superior to that

392 obtained during the other phases. The PN/PS ratio also varied with the HRTs. In phase I,

393 the PN/PS ratio of the extracted EPS was 1.5, which then reached a minimum value of

394 1.3 in phase IV and increased to a maximum value of 2.2 in phase VII. Both the PN

395 portion in the EPS content and the PN/PS ratio had peak values during phase IV. The

396 PN/PS of EPS could play a role in the formation and stabilization of granular sludge. Pol

397 et al. (2004) suggested a high proportion of PN favors sludge granulation because of its

398 high content of negatively charged amino acids, and Basuvaraj et al. (2015) reported the

399 PN/PS ratio should be approximately 1.4 for good settling granular sludge, which

400 supported the observation of the high average settling velocities of granular sludge during

401 phases III–V in this study. The highest PN/PS ratio (2.2) for phase VII, which

402 corresponded to the lowest average velocity and the greatest portion of small particles (≤

403 1 mm), was caused by the decrease in the PS portion of the EPS. As PN predominantly

404 localized in the core region of the granules (Lu et al., 2017; Pol et al., 2004), the PS were

405 easily loosened from the EPS under a high hydraulic upflow velocity (HRT = 2 h), which

406 had a negative effect on sludge granulation (Basuvaraj et al., 2015; Kim et al., 2014).

17
407 Thus, based on overall performance and granular properties, an HRT within the range of

408 12 h to 4.5 h is proposed for MSG wastewater treatment using a UASB reactor.

409

410 3.5 Microbial community analyses

411 3.5.1 Alpha diversity analysis of microbial communities

412 Based on the analysis by 16S rDNA gene sequencing for the sludge samples collected

413 from the UASB reactor during phases I and VII, the Chao1 and ACE estimators, as well

414 as the Simpson and Shannon indices, were calculated and are listed in Table 2 and the

415 Supplementary data. Both the Chao1 and ACE estimators for archaea and bacteria

416 showed a decrease in species richness (Feranchuk et al., 2018) after 141 days of operation.

417 From the decreased Shannon and Simpson indices of both the archaea and bacteria, it was

418 apparent that the diversity of species in the microbial communities in the UASB reactor

419 decreased over time. The results suggested that the operational HRT had a substantial

420 effect on the microbial diversity in the UASB reactor, which is similar to the previous

421 study (Chen et al., 2020).

422

423 Table 2

424

425 3.5.2 Changes in the archaeal community

426 The relative abundance of dominant archaeal populations in the granular sludge samples

427 collected during phases I and VII are shown in Fig. 6 and the Supplementary data. During

428 phase I, the largest genus group in the granular sludge that was also examined in the

429 previous study (Chen et al., 2020) was Methanobacterium, which accounted for a total

430 relative abundance of 59.8% and included the species Methanobacterium beijingense,

18
431 Methanobacterium subterraneum, and Methanobacterium formicicum; these are

432 considered hydrogenotrophic methanogens (Yashiro et al., 2011). The second largest

433 genus group was Methanosaeta, which accounted for a total relative abundance of 34.4%

434 and included Methanosaeta concilii and Methanosaeta harundinacea; these are

435 considered acetoclastic methanogens (Chen et al., 2018b; Fang & Zhang, 2015; Tian et

436 al., 2015). The Methanosarcina genus showed a relative abundance of 4.92% and

437 included Methanosarcina mazei, which is of great ecological importance as it is the only

438 known organism capable of fermenting acetate, methylamines, and methanol to CH4, CO2,

439 and NH3 (in the case of methylamines) (Chen et al., 2018b; Deppenmeier et al., 2002). In

440 contrast, Methanosaeta, which mainly contained the species Methanosaeta concilii,

441 showed a dominant abundance of 77.01% during phase VII. Meanwhile, the relative

442 abundance of Methanobacterium, which included Methanobacterium beijingense,

443 Methanobacterium formocicum, and Methanobacterium subterraneum, decreased to

444 21.87%. Moreover, the relative abundance of Methanosarcina (containing

445 Methanosarcina mazei and Methanosaeta harundinacea) decreased to 0.88%.

446

447 Fig. 6

448

449 Unlike the archaeal community during phase I (where both acetoclastic and

450 hydrogenotrophic methanogens were the primary microbial sequences), the acetoclastic

451 methanogen (Methanosaeta containing Methanosaeta concilii) was dominant and mainly

452 responsible for methane production. The greatly increased population of Methanosaeta

453 and the decrease in hydrogenotrophic methanogens reflected the changes in the

454 intermediate products (Chen et al., 2018b; Lu et al., 2017) during the glutamate

19
455 fermentation process, which supported the excessive accumulation of acetate during

456 phase VII (Fig. 3d). From the dynamic changes in the archaeal community in the UASB

457 reactor, it was apparent that the operational HRT greatly influenced the archaeal

458 community during the long operational period.

459

460 3.5.3 Changes in the bacterial community

461 The relative abundance of dominant bacterial populations in the granular sludge samples

462 collected from phases I and VII are shown in Fig. 6 and the Supplementary data. Eight

463 known bacteria phyla (Firmicutes, Actinobacteria, Synergistetes, Thermotogae,

464 Proteobacteria, Chloroflexi, and Nitrospirae) were identified in the granular sludge

465 sampled during phase I. The dominant genera were determined in ascending relative

466 abundance as follows: Pseudomonas (2.05%), Romboutsia (5.52%), Mesotoga (5.74%),

467 Aminivibrio (8.52%), Clostridium (10.23%), and Actinomyces (22.56%). In contrast, only

468 five known bacterial phyla (Fimicutes, Synergistetes, Proteobacteria, Bacteroidetes, and

469 Saccharibacteria TM7) were identified from the sludge samples collected during phase

470 VII. Within the Firmicutes phylum, Clostridium was the largest genus with a relative

471 abundance of 30.33%, and it included Clostridium pascui, which was found as a

472 glutamate-fermenting spore formerly isolated from the soil samples (Buckel, 2001; Chen

473 et al., 2018b; Wilde et al., 1997). The other genera were ranked in ascending relative

474 abundance (>1%) as follows: Acidaminococcus (1.06%), Klebsiella (1.22%),

475 Saccharimonas (1.24%), Acetoanaerobium (1.91%), Syntrophobacter sp. (4.77%),

476 Syntrophomonas (5.02%), Lentimicrobium (6.94%), and Anaeromusa (9.85%).

477

478 As the largest bacterial phylum in the sludge sample from phase I, Actinobacteria

20
479 disappeared during phase VII and were replaced by Firmicutes (relative abundance of

480 48.17%). The anaerobic environment in the UASB reactor was quite inferior for

481 Actinobacteria (Gupta et al., 2014). Under continuous feeding with glutamate,

482 Clostridium easily adapted to the selective pressure in the UASB reactor under an

483 operational HRT of 2 h, as well as Anaeromusa, which can utilize glutamate to ferment

484 acetate and propionate (Buckel & Thauer, 2013; Strompl et al., 1999). Several diversified

485 VFA degraders were also reserved in the reactor, including Syntrophobacter and

486 Syntrophomonas that degrades propionate or butyrate only in coculture with a H2-using

487 organism; for example, Syntrophobacter wolinii, Syntrophobacter pfennigii, and

488 Syntrophobacter fumaroxidans are involved in the degradation of propionate (Fang &

489 Zhang, 2015; Hatamoto et al., 2007). The substrate (glutamate) and its intermediates

490 (acetate, propionate, butyrate, and valerate) provided a strong selective pressure that

491 drove bacterial evolution in the UASB reactor (Figs. 3 and 6). Therefore, the results of

492 the bacterial community analysis agree well with the experimental data.

493

494 3.6 Biodegradation pathways of glutamate in the UASB reactor

495 To investigate the biodegradation pathways of glutamate (Liang et al., 2007; Lu et al.,

496 2017), SMA tests were conducted on samples taken during each operational phase (I–VI)

497 by feeding them methanol, acetate, propionate, butyrate, valerate, glutamate, and H2/CO2,

498 respectively. The corresponding Rmax and SMA values, which reflected the utilization

499 ability of biomass for a specific substrate, are shown in Table 3. Both Rmax and the SMA

500 changed with the operational phases. For the substrates used, both acetate and glutamate

501 caused obvious increases in the SMA values, whereas the methanol and H2/CO2 reflected

502 inflexible values. It is remarkable that a large amount of VFAs accumulated in phase VII,

21
503 which could attributed to the HRT decrease or the doubled organic loading rate. Under a

504 short HRT of 3 h (phase VI), the methane-producing capacity (Fang & Zhang, 2015; Kim

505 et al., 2014) of the granular sludge, with the tested substrates in descending order, was as

506 follows: acetate > glutamate > butyrate > H2/CO2 > valerate > propionate.

507

508 Table 3

509

510 A sharp increase in the SMA for the sludge fed with acetate (Table 3) could be related to

511 the increase in the number of Methanosaeta (Supplementary data). An abundance of

512 hydrogenotrophic methanogens, such as Methanobacterium bei

513 jingense, Methanobacterium subterraneum, and Methanobacterium formicicum,

514 remained during phase VII and maintained a steady value in the SMA fed with H2/CO2.

515 Bacterial degraders for glutamate fermentation, including Clostridium pascui and

516 Anaeroarcus burkinensis (Supplementary data), increased in population with the

517 operational HRTs. Thus, compared to phase II, the methane-producing capacity of the

518 granular sludge for glutamate increased more than seven times during phase VI (Table 3).

519 The high utilization ability of butyrate in the SMA test during phase VI can likely be

520 attributed to the large population of Syntrophomonas sp. that appeared in the UASB

521 reactor after a long operational period.

522

523 The most common species in the bacterial domain during phase VII was Clostridium

524 pascui (Supplementary data), which is classified in Clostridium cluster I (Wilde et al.,

525 1997) and can utilize (S)-glutamate via the 3-methylaspartate pathway with fermentation

526 products of ammonium, acetate, butyrate, hydrogen, and carbon dioxide according to Eq.

22
527 (4) (Buckel, 2001; Buckel & Thauer, 2013).

528

529 (4)

530 The main biodegradation pathway via butyrate production is favorable over propionate

531 oxidization, as the latter pathway via propionate oxidization has higher activation energy

532 barriers (Stams & Plugge, 2009). In addition, glutamate can be converted to acetate and

533 propionate (and traces of H2/CO2) by Anaeroarcus burkinensis (Strompl et al., 1999),

534 which was identified as the second most common species with a relative abundance of

535 9.85% (one-third times that of Clostridium pascui) during phase VII (Supplementary

536 data). Acidaminococcus sp., with a small relative abundance (1.06%), can also

537 decompose glutamate via the 2-hydroxyglutarate pathway into butyrate, propionate,

538 acetate, and H2/CO2 (Buckel, 2001; Kim et al., 2004).

539

540 Based on the SMA tests and the analysis of dynamic evolution of a microbial community,

541 probable pathways of glutamate degradation and methanogenesis for the glutamate-rich

542 wastewater treatment in the UASB reactor are summarized in Fig. 7.

543

544 Fig. 7

545

546 Although at least three pathways for glutamate degradation occurred in the anaerobic

547 reactor, the pathway via 3-methlaspartate by Clostridium pascui was dominant in the

548 UASB reactor (HRT = 2 h). From there, VFAs were converted into acetate and H2 by

549 Syntrophomonas sp., Acidaminococcus sp., and Syntrophobacter sp. Finally, acetoclastic

550 and hydrogenotrophic methanogens were responsible for methane production, among

23
551 which Methanosaeta was dominant under a short operational HRT. Overall, in the UASB

552 reactor for glutamate-rich wastewater treatment during the long operational period, the

553 anaerobes converted glutamate into butyrate or propionate, then to acetate and H2/CO2,

554 and then again for methane production. Additional research seeking new evidence to

555 support these pathways is necessary for future wastewater treatment.

556

557 4. Conclusions

558 The UASB reactor showed good potential for glutamate-rich wastewater treatment. At

559 HRTs of 4.5 h to 6 h, a high and stable COD removal efficiency of more than 95% with a

560 methane yield of 0.31 L-CH4/g-COD was obtained, as well as advantageous properties of

561 granular sludge, including granular sludge diameter, settling velocity, and EPS content.

562 At a short HRT of 2 h, excessive VFAs accumulated, which led to a deterioration in

563 reactor performance. At least three degradation pathways occurred for glutamate

564 fermentation in the UASB reactor, and the pathway via 3-methlaspartate by Clostridium

565 pascui was likely dominant for glutamate-rich wastewater treatment at an HRT of 2 h.

566

567

568 Acknowledgements

569 This work was supported by the National Natural Science Foundation of China (Grant No.

570 51308068) and the China Hunan Provincial Science & Technology Department (Grant

571 No. 2017SK2361). The authors gratefully acknowledge the support from Professor

572 Yu-You Li (Tohoku University, Japan), the Japan Society for the Promotion of Science,

573 and the Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). We would like to

574 thank Editage (www.editage.cn) for English language editing.

24
575

576 Supplementary data

577 E-supplementary data for this article can be found online.

578

579

25
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737 https://doi.org/10.1016/j.biortech.2007.04.046

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32
748 whole-genome assemblies. Int. J Syst. Evol. Micr. 67, 1613.

749 https://doi.org/10.1099/ijsem.0.001755

750

33
751 Figure Captions

752 Fig. 1 Schematic diagram of the experimental apparatus

753 Fig. 2 Overall performance of the UASB reactor in treating synthetic MSG wastewater

754 under various HRTs: (a) pH; (b) NH4+-N; (c) alkalinity; (d) COD; (e) volatile fatty acids

755 (VFAs); (f) biogas production and methane content

756 Fig. 3 Effects of HRT on MSG wastewater removal: (a) NH4+-N production; (b) VFA

757 concentration; (c) organic removal rate (ORR) and COD removal efficiency; (d) methane

758 yield and methane production rate (MPR)

759 Fig. 4 COD mass balance in the UASB system

760 Fig. 5 Characteristics of granular sludge

761 Fig. 6 Microbial community structure during phases I and VII

762 Fig. 7 Proposed pathways of methanogenic degradation of glutamate in the UASB

763 reactor

34
Table 1 Operational conditions of the UASB reactor.

Phase I Phase II Phase III Phase IV Phase V Phase VI Phase VII

Parameter Days Days Days Days Days Days Days

1-35 36-63 64-82 83-106 107-127 128-149 150-180

1916.52 2109.05 2032.52 2060.72 2054.25 2043.45 2032.38

COD (mg/L)
± 251.50 ± 114.20 ± 121.48 ± 129.23 ±100.16 ± 109.45 ± 131.02

HRT (h) 48 24 12 6 4.5 3 2

OLR (g-COD/L/d) 0.95±0.13 2.08±0.11 3.98±0.24 8.26±0.52 10.82±0.50 16.20±0.88 24.66±1.57

Influent flow (L/d) 3 6 12 24 32 48 72

Temperature ( ) 35±1

pH of influent 7.0±0.5
Table 2 Richness and diversity estimation for the microbial community in the UASB reactor during each phase.

Community richness estimators Community diversity estimators

Samples
ACE Chao1 Shannon Simpson

Phase I 19 19 1.49 0.29

Archaea
Phase VII 15 16 0.81 0.61

Phase I 382 380 3.72 0.07

Bacteria
Phase VII 346 352 3.42 0.11
Table 3 The SMA and maximum methane production rate (Rmax) of the granular sludges with different substrates.

Parameters Rmax (mL-CH4/h) SMA (g-COD CH4/g VSS/d)

Operational phase/HRT I/48 h II/24 h III/12 h IV/6 h VI/3 h I/48 h II/24 h III/12 h IV/6 h VI/3 h

Methanol 2.02 1.89 2.04 1.67 N.D.* 0.044 0.040 0.041 0.028 0.003

Acetate 0.38 0.90 1.42 2.07 3.55 0.008 0.019 0.028 0.034 0.101

Propionate 0.53 0.36 0.20 0.16 0.15 0.012 0.008 0.004 0.003 0.004

n-Butyrate N.D.* N.D.* N.D.* N.D.* 2.23 N.D.* N.D.* N.D.* N.D.* 0.064

n-Valerate N.D.* N.D.* N.D.* N.D.* 0.23 N.D.* N.D.* N.D.* N.D.* 0.007

(S)-glutamate N.D.* 0.56 0.87 1.75 2.90 N.D.* 0.011 0.017 0.029 0.083

H2/CO2** 0.74 1.93 1.56 1.59 1.45 0.016 0.041 0.031 0.026 0.041

*
N.D.: not determined.

**
H2/CO2: the mixed gas consisted of H2 and CO2 (80:20, v/v).
 Effluent
Gas Meter
Dryer /
Buffer Bottle Desulfurizer

Thermometer
Water Bath
Biogas
Collector
Pump

Thermostat
Sampling Port

Influent

Up-flow Anaerobic Sludge Substrate Tank

Blanket (UASB)
 P h a se s
9 Ⅰ Ⅱ
4 Ⅲ Ⅳ Ⅴ Ⅴ I Ⅶ P h a se s Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ Ⅶ

( )
3
b In f.
In f. a
8
E ff.

m g /L
E ff.

2
1 0
7
p H

A m m o n iu m
6 0 100
)

3 c d
80
m g -C a C O 3 /L

In f. to ta l a lk a lin ity

( )
3

R e m o v a l E ffic ie n c y (% )
In f. b io c a r b o n a te a lk a lin ity

2
E ff. to ta l a lk a lin ity
60
1 0 3 m g /L
E ff. b io c a r b o n a te a lk a lin ity

40
2

1
( 3

In f.

20
1 0

C O D 1 E ff.
A lk a lin ity

R e m o v a l e f f ic ie n c y

0 1
00 0
12 f 1 0
0
M e th a n e p e r c e n ta g e in b io g a s (% )

80
e

B io g a s p r o d u c tio n r a te (L /L /d )
M e th a n e p e rc e n ta g e

8
V a le r ic a c id

9
( )

B u ty r ic a c id B io g a s p r o d u c tio n r a te

60 6
P r o p io n ic a c id
m g /L

6
A c e tic a c id

40 4
2
1 0

3 20 2
V F A

0020406080100120140160180 00204060801001201401601800
O p e r a tio n D a y s (d ) O p e r a tio n D a y s (d )
 100
60 VSS a
VSS/TSS 80

VSS/TSS (%)
VSS (g/L)

40 60

40
20
20

0 0

Average settling velocity (m/h)

>3 mm 2~3 mm 1~2 mm
b
Particle size portion (%)

120
0.5~1 mm <0.5 mm
150
Average settling velocity
90

100
60

50
30

0 0
5
250 PN
PS
c
4
EPS content (mg/g)

200 PN/PS

PN/PS ratio
3
150

100 2

50 1

0 0

Phases I II III IV V VI VII

 Euryarchaeota Euryarchaeota
Archaea

Archaea
Methanobacterium Methanosaeta
Methanosaeta Methanobacterium
Methanosarcina Methanosarcina
others others
Actinobacteria Firmicutes
Actinomyces Clostridium
Firmicutes Anaeromusa
Clostridium Syntrophomonas
Romboutsia Acetoanaerobium
Synergistetes Acidaminococcus
Bacteria

Bacteria
Aminivibrio Bacteroidetes
AQRZ_g Lentimicrobium
Thermotogae DQ677001_g
Mesotoga Proteobacteria
Chloroflexi Syntrophobacter
AF423186_g Actinobacteria Firmicutes Klebsiella
Firmicutes
Nitrospirae Synergistetes
Synergistetes
Proteobacteria
Synergistetes
AB262729_g Proteobacteria Bacteroidetes AF280863_g
Thermotogae
Proteobacteria Chloroflexi
Saccharibacteria TM7 Saccharibacteria_TM7
Pseudomonas others Saccharimonas
Nitrospirae
others others others
0 10 20 30 40 50 60 70 80 70 60 50 40 30 20 10 0
Phase Ⅰ Percentage (%) Percentage (%) Phase Ⅶ
 (S)-Glutamate acid

Clostridium pascui
Anaeroarcus burkinensis
(s)-Citramalate
Acidaminococcus sp.

Pyruvate Propionate

Syntrophobacter sp.
Butyrate

Syntrophomonas sp.
H2/CO2
Acetoanaerobium sp.

Acetate
Methanobacterium beijingense
Methanosaeta concilii Methanobacterium subterraneum
Methanobacterium formicicum
CH4
 Highlights

• Methanogenic degradation of glutamate in UASB reactor depended on HRT

applied

• High MSG removal and energy recovery obtained at HRTs of 4.5–6 h

• Clostridium and Methanosaeta were predominant in glutamate-degrading

granules

• HRT had substantial impact on SMA and microbial community of granules

• The pathway via 3-methlaspartate was dominant for methanogenic

degradation
 Author Contribution Statement

of “Anaerobic treatment of glutamate-rich wastewater in a continuous UASB

reactor: Effect of hydraulic retention time and methanogenic degradation

pathway” (Reference No.: CHEM66161)

by H. Chen, et al.

Hong Chen: Conceptualization, Methodology, Project administration, Funding acquisition

Yanxiao Wei: Methodology, Data curation, Writing- Original draft preparation, Validation

Chenglei Xie: Investigation, Writing - original draft

Hong Wang: Methodology, Writing- Original draft preparation

Sheng Chang: Resources, Writing- Original draft preparation

Ying Xiong: Resources, Validation, Writing- Reviewing and Editing

Chunyan Du: Resources, Funding acquisition, Validation

Benyi Xiao: Funding acquisition, Validation, Supervision

Guanlong Yu: Resources, Funding acquisition, Formal analysis

 Declaration of Interest Statement

Article Title: Anaerobic treatment of glutamate-rich wastewater in a continuous UASB reactor:

Effect of hydraulic retention time and methanogenic degradation pathway

Authors: Hong Chen, Yanxiao Wei, Chenglei Xie, Hong Wang, Sheng Chang, Ying Xiong,

Chunyan Du, Benyi Xiao*, Guanlong Yu

All authors declare that we have no financial and personal relationships with other people or

organizations that can inappropriately influence our work. We confirm that this manuscript

has not been published elsewhere and was not previously submitted to Chemosphere. All

authors have approved the manuscript and agree with its submission to Chemosphere. All

authors of this manuscript have directly participated in the planning, execution, and analyses

of this study.

Signature: Benyi Xiao (on behalf of all co-authors of this manuscript)

Date: September 25, 2019

Institution: Research Center for Eco-Environmental Sciences, Chinese Academy of

Sciences (Corresponding author: B.Y. Xiao*)