Hydrogen Recovery from Waste Activated Sludge: Role of Free

Subscriber access provided by READING UNIV

Article

Hydrogen recovery from waste activated sludge: Role of free nitrous acid (FNA) in a pre-fermentation-microbial electrolysis cells system Zhihong Liu, Aijuan Zhou, Jiaguang Zhang, Sufang Wang, Yunbo Luan, Wenzong Liu, Aijie Wang, and Xiuping Yue ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04201 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on February 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Sustainable Chemistry & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

1

Hydrogen recovery from waste activated sludge: Role of free nitrous

2

acid (FNA) in a pre-fermentation-microbial electrolysis cells system

3

Zhihong Liu 1, Aijuan Zhou 1, 2, *, Jiaguang Zhang 3, Sufang Wang 1, Yunbo Luan 4,

4

Wenzong Liu 5, Aijie Wang 5, 6 and Xiuping Yue 1, 2, *

5

1

Technology, 79 Yingzexi Road, Taiyuan 030024, P.R. China

6 7

2

3

4

5

Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, Haidian District, Beijing 10085, P.R. China

14 15

College of Mechanics, Taiyuan University of Technology, 79 Yingzexi Road, Taiyuan 030024, P.R. China

12 13

College of Architecture and Civil Engineering, Taiyuan University of Technology, 79 Yingzexi Road, Taiyuan 030024, P.R. China

10 11

Shanxi Engineer Research Institute of Sludge Disposition and Resources, Taiyuan University of Technology, 79 Yingzexi Road, Taiyuan 030024, P.R. China

8 9

College of Environmental Science and Engineering, Taiyuan University of

6

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute

16

of Technology (SKLUWRE, HIT), 73 Huanghe Road, Nangang District, Harbin

17

150090, P.R. China

18 19

*Corresponding author:

20

College of Environmental Science and Engineering, Taiyuan University of

21

Technology, 79 Yingzexi Road, Taiyuan 030024, Shanxi Province, P.R. China

22

E-mail: [email protected] (A. Zhou); [email protected] (X. Yue);

23

Tel./fax: +86 0351-3176581.

24 25 26 1

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

27

Abstract

28

Due to the limited hydrolysis rate of particulate organics and suitable substrates for

29

hydrogen-producing bacteria in raw waste activated sludge (WAS), traditional

30

fermentative hydrogen production has low hydrogen yield and energy recovery

31

efficiency. The role of free nitrous acid (FNA) pretreatment on WAS and hydrogen

32

recovery was investigated in a pre-fermentation-microbial electrolysis cells (MECs)

33

system. The results demonstrated that WAS hydrolysis and acidification were

34

enhanced by FNA pretreatment. Notably, the accumulation of acetic acid and

35

propionic acid eventually reached to 55% and 22% during pre-fermentation. During

36

MECs cascade utilization, volatile fatty acids (VFAs) were exhausted and the

37

utilization efficiencies of soluble carbohydrates and proteins reached 62% and 41.5%,

38

respectively. The hydrogen yield from FNA-pretreated sludge was 1.44 mL/g VSS,

39

which was approximately 3 times than that of the control. High-throughput

40

sequencing and canonical correspondence analysis revealed that FNA pretreatment

41

promoted the hydrolysis and acidification of particulate organics through

42

accumulating anaerobic fermentation bacteria (AFB) in pre-fermentation, furthermore,

43

stimulated the increase of electrochemically active bacteria (EAB) and therefore

44

enhanced the current and hydrogen production. This study may provide a sound basis

45

for the potential implementation of FNA pretreatment to accomplish organics

46

cascading utilization and the synchronous recovery of energy from WAS.

47

Keywords

48

Waste activated sludge (WAS); Free nitrous acid (FNA); Anaerobic pre-fermentation;

49

Microbial electrolysis cells (MECs); Hydrogen production

50 51

2


Page 2 of 29

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


52 53

Introduction Waste activated sludge (WAS) is the most voluminous by-product of biological

54

wastewater treatment, which requires proper handling, treatment and disposal.1-4 In

55

China, 30~40 million tons of WAS (80% moisture content) are produced annually, and

56

the amount is expected to reach 60~90 million tons by 2020. WAS is considered to be

57

a potential source of organic compounds, containing large amounts of carbohydrates

58

and proteins, which account for 35-61% and 7-11% of the total chemical oxygen

59

demand (TCOD) of WAS.5 Relevant research has concentrated on transforming these

60

considerable organics into biogas, fertilizer, or easily-biodegradable carbon sources.6

61

Recently, greater attention has been paid to bio-hydrogen production from WAS due

62

to its high energy yield of 142.9 KJ/g and zero pollution in utilization.7

63

Unfortunately, most biodegradable organics in WAS is either enclosed inside the

64

microbial cell wall or enmeshed in extracellular polymeric matrix, which further

65

contributes to limit the biodegradability of these organics into hydrogen. In order to

66

accelerate the hydrolysis of organic matters, WAS is often pretreated by various

67

physical and chemical methods prior to test.8 Lately, free nitrous acid (FNA),

68

protonated form of nitrite, was demonstrated to be an effective and promising method

69

for WAS pretreatment.9-11 It was found that FNA increased biodegradability by

70

generating multiple inhibitory effects on microbial metabolism of extensive

71

microorganisms,12 being extraordinarily biocidal to microorganisms at parts per

72

million (ppm) or even sub-ppm levels.13-14 FNA pretreatment was reported to be

73

effective on enhancing hydrolysis rate, volatile fatty acids (VFAs) and methane

74

production of WAS. The biodegradability of FNA-pretreated sludge could be

75

promoted by 20%-50% based on biochemical methane potential (BMP) testing.10, 15-16

76

Methane production could be improved by 10-30% at FNA concentrations in the 3



77

range of 0.4-2.1 mg N/L for 24 h.10 Additionally, Li et al. (2016) proved the shorter

78

fermentation time and highest VFAs production could achieve at 1.8 mg N/L of

79

FNA.17 However, an improvement of the biogas production rate is further considered

80

by upgrading conventional AD systems.18-19

81

MECs is a prospective technology for hydrogen production via the utilization of

82

substrates from WAS. Recently, a number of studies have suggested that combine

83

MECs with anaerobic digestion (AD), could not only increase the positive synergisms

84

established in the digesters, but also provide an utilization route for the existing

85

digestate, which further accomplish the bio-hydrogen recovery from WAS.7 Wang et

86

al. (2014) obtained the yield of hydrogen at 8.5 mg H2 g-1 VSS feeding with sodium

87

dodecylsulphate (SDS)-treated WAS fermentation liquid (SFL).5 Zhou et al. (2016)

88

harvest 12.90 mg H2 g-1 VSS from Rhamnolipid bio-surfactant (RL)-treated SFL.20

89

In this study, the feasibility of hydrogen recovery and cascade utilization of

90

organics from WAS were investigated in a pre-fermentation-microbial electrolysis

91

system. The performance of FNA pretreatment on WAS solubilization and

92

acidification during the pre-fermentation process was studied. The hydrogen

93

production and the degradation rules of the substrates of the fermented

94

FNA-pretreated WAS were analyzed during the subsequent MECs process.

95

Furthermore, high-throughput sequencing analysis was used to evaluate the

96

mechanism of FNA pretreatment for enhancing hydrogen recovery from WAS on the

97

microorganism level. In addition, we used canonical correspondence analysis (CCA)

98

to assess the correlations between major environmental variables and functional

99

microbial populations.

100 101

4


Page 4 of 29

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


102

Material and methods

103

FNA pretreatment and pre-fermentation of WAS

104

WAS was collected from a Zhengyang municipal wastewater treatment plant

105

(WWTP) in Jinzhong City, China. Prior to use, WAS was concentrated by settling for

106

24 h at 4 °C, and the supernatant was then removed. To prevent clogging problems,

107

concentrated WAS was screened with a 40-mesh sieve. The main characteristics

108

(average value, plus standard deviation of triplicates) were as follows: total suspended

109

solids (TSS): 26.4 ± 0.4 g/L; volatile suspended solids (VSS): 12.1 ± 0.2 g/L; total

110

chemical oxygen demand (TCOD): 26.2 ± 0.2 g/L; soluble chemical oxygen demand

111

(SCOD): 0.41 ± 0.05 g/L; VFAs: 255 ± 12 mg COD/L; soluble carbohydrates: 44.56 ±

112

8 mg COD/L; and soluble proteins: 37.5 ± 2 mg/L, pH 7.03 ± 0.02.

113

Three serum bottles with a working volume of 400 mL each were set-up for

114

WAS pretreatment by FNA. This process was termed “Phase 1”. A nitrite stock

115

solution was first added to reactors to achieve a NO2--N concentration of 300 mg N/L.

116

The pH was adjusted to 5.5 ± 0.1 by adding 3.0 M HCl. Twenty milliliters of

117

phosphate buffer solution (PBS) was added in each reactor to maintain moderately

118

acidic conditions. The pretreatment lasted for 24 h, during which the temperature was

119

maintained at 22 ± 1 °C. Under these conditions, the FNA concentration was titrated

120

to 2.13 mg N/L based on a calculation applying the formulae SNO2--N/(Ka×10pH) and

121

Ka=e-2300/(273+T) 21, which proved to be an optimal FNA dosage for WAS

122

solubilization.10 To evaluate the effect of FNA pretreatment, NO2--N, NO3--N, NH4+-N,

123

soluble proteins and soluble carbohydrates were measured every 4 hours.

124

Anaerobic pre-fermentation experiments were carried out in six batch reactors, a

125

process that was termed “Phase 2”. These reactors, with a 400-mL working volume

126

each, were divided into two groups (every three reactors were replicates for each 5



127

group). One group was fed concentrated WAS (hereafter referred to as the

128

AD_Control test). The feedstock for the other group was FNA-pretreated WAS

129

(hereafter referred to as the AD_FNA test). Some of the fresh sludge was maintained

130

under aeration at room temperature overnight and then used as inoculum with an

131

addition ratio of 1:9 (v/v). Nitrogen gas was pumped into reactors for 10~15 min in

132

order to remove oxygen; then, all reactors were capped, sealed, and stirred in an

133

air-bath shaker (120 rpm) at 35 ± 1 °C.

134

MECs reactors setup and operation

135

Ten single-chamber MECs reactors were constructed simultaneously and

136

inoculated using aeration tank effluent from the same WWTP in Jinzhong. Details of

137

volume, anode and cathode were provided previously.7, 22 The reactors were operated

138

at room temperature (22 ± 1 °C), a voltage of 0.80 ± 0.01 V was applied (Switching

139

Power Supply, FDPS-150, Fudantianxin Inc. China), and 100 mM PBS (pH 7.0) and

140

1,500 mg/L acetate were prepared for the feedstock of the MECs set-up. The setup

141

time lasted for 10 days in approximately 24-h batches until the currents reached 2 mA,

142

and the coulombic efficiencies were greater than 90%. Then, six parallel reactors were

143

selected for the subsequent experiment. Reactors were divided into two groups in a

144

process termed “Phase 3”. The pre-fermented WAS was added to each group, mixed

145

with 100 mM PBS (VWAS:VPBS=1:1) and incubated for 3 days as a cycle to make full

146

use of the organics.

147

DNA extraction and Illumina MiSeq sequencing

148

Illumina MiSeq sequencing was used to analyze the diversity of the microbial

149

community. Before DNA extraction, sludge samples of pre-fermentation experiments

150

and carbon brushes (bio-anode) were centrifuged at 8000 × g to remove the

151

supernatant. DNA was extracted in triplicate from sludge sediments using an EZNA® 6


Page 6 of 29

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


152

Soil DNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA) and subsequently pooled

153

together. Based on the amplification of V3-V4 region of the 16S rRNA gene, the

154

commonly used PCR primers 341F (5′-CCCTACACGACGCTCTTCCGATCTG-3′)

155

and 805R (5′-GACTGGAGTTCCTTGGCACCCGAGA ATTCCA-3′) were selected.

156

During sequencing, sample multiplexing was achieved by means of the incorporation

157

of barcodes between the forward primer and 454 adaptor. Polymerase chain reactions

158

(PCRs) were performed as described previously.23-24 Then, the PCR amplicon was

159

purified and quantified and was further applied to sequencing on an Illumina MiSeq.

160

For taxonomic analysis, the 16S rRNA gene sequences read was performed

161

individually using Ribosomal Database Project (RDP) classifier. Raw sequence data

162

of the study were deposited to the NCBI Short Read Archive database with the

163

accession no. SRR5482157. Notably, we trimmed the adapters, barcodes, and primers

164

in all raw sequences to avoid the effects of random sequencing errors. The sequence

165

was removed if it was shorter than 350 bp or contained any ambiguous base cells.

166

The remaining sequences were clustered into operational taxonomic units

167

(OTUs) using a 97% identity threshold (3% dissimilarity level). Rarefaction curves

168

were generated and alpha diversity measurements, including the Shannon index

169

(http://www.mothur.org/wiki/Shannon), Chao1 index

170

(http://www.mothur.org/wiki/Chao) and ACE index

171

(http://www.mothur.org/wiki/Ace), were calculated for each sample. BLAST of

172

taxonomic classification was conducted using MOTHUR program via SILVA database

173

with a set confidence threshold of 80%. Hierarchical clustering analysis (HCA) was

174

used to display the interrelationship among the four samples intuitively on the basis of

175

the depths of colors, which represented the abundance of the bacteria communities. In

176

this study, we performed HCA analysis using GraPhlAn 0.9.7 and iTOL 3.2.1. 7



177

Cytoscape v3.2.1 was used to generate OTUs networks, which could further delineate

178

the similarity and diversity between the different sludge samples. The central portion

179

was shared OTU, while the species and numbers of the other bacterium showed the

180

diversity among the four samples.25 Principal coordinates analysis (PCoA) visually

181

reflected beta diversity, which was calculated using the distance matrices based on the

182

UniFrac phylogenetic method. Canonical correspondence analyses (CCA) were

183

generated by Canoco 4.5 to evaluate the specific correlations between characteristic

184

genera and environmental factors measured in the study, including methane, VFAs,

185

NO2--N, current, hydrogen, soluble proteins and carbohydrate concentrations. The

186

relative abundance of 16 characteristic bacteria was used in the CCA analysis.

187

Analysis and calculation methods

188

WAS samples were centrifuged at 10,000 × g for 10 min, filtered through a

189

0.45-µm cellulose nitrate membrane filter and stored at 4 °C prior to analysis. TCOD,

190

SCOD, TSS, VSS, NH4-N, NO3--N and NO2--N were measured according to standard

191

methods (APHA, 1998). Soluble proteins were measured using a bicinchoninic acid

192

(BCA) protein quantitation kit, whereas soluble carbohydrates was determined using

193

the phenol–sulfuric acid method with glucose as the standard.26 VFAs were analyzed

194

using gas chromatography, and the total concentration comprised acetic acid (HAc),

195

propionic acid (HPr), n-/iso- butyric acid (n-HBu/iso-HBu) and n-/iso- valeric acid

196

(n-HVa/iso-HVa), expressed as the COD concentration (mg COD/L). The equivalent

197

relationships between COD and various VFAs were as follows: 1.07 g-COD/g-HAc,

198

1.51 g-COD/g-HPr, 1.82 g-COD/g- (n-/iso-) HBu, and 2.04 g-COD/g- (n-/iso-) HVa.

199

The gases produced by anaerobic reactors and MECs were collected in gas bags, and

200

the total volumes were measured by glass syringe. Gas composition was analyzed by

201

gas chromatography. The voltage and current were recorded via a data recorder 8


Page 8 of 29

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


202

(model 2700, Keithley Instrument, USA) every 10 minutes. Crucial indices to judge

203

the performance of MEC reactors included coulombic efficiency (CE), current density

204

(A/m2, normalized to the projected cathode area), hydrogen production rate Q

205

(m3-H2/m3/day), hydrogen yield YH2 (mL H2/g VSS) and energy recovery efficiency.27

206

Energy efficiency (ηE, %) was calculated on the basis of electricity input as ηE = WH2 /

207

Win, where WH2 is the energy content of hydrogen based on the heat of combustion

208

(upper heating value of 285.83 kJ per mole of H2) and Win is the electricity input

209

determined as ܹ௜௡ = ‫ܧܫ(׬‬௔௣ − ‫ܫ‬ଶ ܴ) ∙ ݀‫ݐ‬, where Eap is the applied voltage (0.8 V in

210

this study) and R is the external resistance (10 Ω) 28.

211

Results and discussion

212

Effect of FNA pretreatment on WAS solubilization and acidification

213

The degree of WAS solubilization can be expressed by the change of soluble

214

protein and carbohydrate levels.29-30 As shown in Fig. 2A and B, soluble carbohydrate

215

and protein levels increased with FNA pretreatment and peaked at 298.7 ± 6.5 mg

216

COD/L and 665.3 ± 30.1 mg COD/L (12 h), which was 6.7 and 18.4 times higher than

217

that of the control, respectively. The eventual values were still much higher than that

218

of un-pretreated WAS. Remarkably, NO2--N concentration increased with treatment

219

time and peaked at 226.9 ± 7.5 mg/L during the initial 12 h. NO3--N content also

220

achieved its maximum (40.4 ± 3.9 mg/L), which demonstrated that the nitration

221

process occurred during Phase 1 (Fig. 1A). No methane was produced but 140 mL of

222

gas was collected at the end of the pretreatment, consisted with N2 and N2O. The

223

results could in accordance with Wang et al. (2013), who detected only N2 on the first

224

day and proved that denitrification occurred during FNA pretreatment.10 In short, the

225

results indicated that FNA pretreatment was effective in enhancing WAS

226

solubilization. 9



227

In 96-h pre-fermentation, the effect of FNA pretreatment on soluble

228

carbohydrates and proteins is shown in Fig. 2A and B. Both soluble carbohydrates and

229

proteins were clearly elevated from 48 h onward and decreased slightly with the time

230

extension. By comparison, soluble proteins fluctuated slightly. The final

231

concentrations of soluble carbohydrates and proteins in the AD_FNA test (120 h)

232

were 2.46 and 4.14 times higher than that of the AD_Control test. Analogously, as a

233

by-product of protein degradation, the trend of ammonia was similar with that of

234

proteins (Fig. 1B). The concentration of VFAs in the AD_FNA test reached its

235

maximum (1761 mg COD/L) on the fourth day, which was 5.0-fold of that obtained in

236

the AD_Control test (Fig. 2C, Phase 2). Obviously, the components of VFAs reflected

237

significant difference. HAc and HPr, the two top individual VFAs, reached 55% and

238

22% in the AD_FNA test, respectively (Fig. 2D). However, the levels in the

239

AD_Control test were 10% and 61%, respectively (Fig. 2D). The n-HVa was the

240

lowest VFAs in any of the measured reactors, which was less than 3%. Thus, FNA

241

pretreatment could expedite the production of VFAs, particularly HAc.

242

Hydrogen recovery and organics utilization during MECs cascade utilization

243

Fig. S1A depicted the current variation during the MECs startup. The average

244

current tended to be stable and eventually decreased from ~7 mA to 2 mA (> 2 mA).

245

Moreover, the coulombic efficiency and hydrogen yield rate were elevated from 85 ±

246

6% to 101 ± 5% and 79 ± 6% to 154 ± 12%, respectively. On basis of the energy of

247

electricity input, the average energy efficiency of hydrogen yield was up to 140 ± 11%

248

at 0.8 V applied voltage. These phenomena implied that the MECs were successfully

249

constructed.

250

During MECs operation, the variation of electron transport (current) was

251

connected with soluble substrates, especially VFAs degradation.5 As shown in Fig. 10


Page 10 of 29

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


252

S1B, FNA pretreatment contributed to abundant soluble substrates for exoelectrogens,

253

accordingly, the maximal current density was 11 A/m2; while the limited releases of

254

soluble substrates in the control group resulted in the lower current density (only 4

255

A/m2). The currents both stepped to decreases along with the consumption of soluble

256

substrates (Fig. 2C). The hydrogen production yield was 1.44 mL/g VSS in the FNA

257

test, which was 2.7-fold higher than that of the control test (Fig. 2E). FNA

258

pretreatment promoted accumulation of HAc and HPr in pre-fermentation (account

259

for 77% of VFAs), which benefited hydrogen production and further shortened

260

operational time.5

261

As seen from Fig. 2A, the soluble carbohydrates began to decline sharply at 126

262

h and the final removal efficiency increased to 62% in the FNA test, whereas in the

263

control test, it was only 43%. The change of ammonia was consistent with soluble

264

proteins (Fig. 1A), which markedly increased at 126 h and then appeared to integrally

265

decrease. However, the eventual removal efficiencies (34.7% and 41.5% in control

266

test and FNA test, respectively) were still less than that of soluble carbohydrates (Fig.

267

2B). These results implied that soluble carbohydrates were much easier to utilize by

268

exoelectrogens than large-molecule proteins.

269

VFAs, one of the main compositions of fermented WAS, could be easily

270

consumed by exoelectrogens,28 as shown in Fig. 2C, reduced acutely in both FNA and

271

control tests; the respective utilization efficiencies were 99% and 83%. It is reported

272

that HAc and HPr are preferred as main electron donors, which provide significant

273

current generation, while the other VFAs (HBu and HVa) mainly contribute to

274

exoelectrogens metabolism.31 Accordingly, all components of VFAs were also

275

consumed maximally (Fig. 2E). The results demonstrated that on the basis of FNA

276

pretreatment, the soluble substrates released in pre-fermentation accomplished the 11



277

cascade utilization in MECs and further achieved higher hydrogen production.

278

Microbial community analysis

279

The bacterial communities of AD_Control and AD_FNA, as well as the biofilm

280

developed on the anodes of MECs, fed with fermented AD_Control sludge

281

(AD_MEC_Control) and AD_FNA sludge (AD_MEC_FNA) were analyzed using

282

Illumina MiSeq sequencing. In total, as shown in Table S1, the number of OTUs in

283

the four bacteria samples was 13,697, of which 542 OTUs (4% of the total OTUs)

284

were shared by all samples. The shared OTUs were mainly grouped into three phyla:

285

Proteobacteria (41.9%), Bacteroidetes (12.7%), and Firmicutes (10.9%) (Fig. 4).

286

Rarefaction curves for all libraries not only illustrated that the sequences of all

287

samples were reasonable but also displayed shapes indicative of eﬀective sampling of

288

the community diversity (Fig. S2A). The microbial diversities of the involved

289

communities in all samples were assessed based on α-diversity. Under FNA

290

pretreatment, the Shannon index of samples decreased from 6.08 (AD_Control) to

291

5.47 (AD_FNA), and further declined to 5.45 (AD_MEC_FNA) in the MECs system

292

(Table S1). Based on Chao1 and ACE indices, which indicate richness, the

293

AD_MEC_FNA sample also had relatively lower diversity (13,493 and 23,228,

294

respectively). β-diversity was used to calculate and examine the similarity of the

295

microbiome. PCoA exhibited the dissimilarity of bacterial community composition

296

among the samples, based on unweighted UniFrac (Fig. S2B). The principal

297

components 1 and 2 were 36.53% and 34.19%, respectively. In addition, the four

298

samples were clearly separated from each other, which illustrated the significant

299

differences in the bacterial community composition (Fig. S2B). This could be further

300

demonstrated by the hierarchical clustering analysis results (Fig. 3D).

301

Phylogenetic differences in 16S rRNA gene sequences were characterized at 12


Page 12 of 29

Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


302

three different level classifications, including that of phylum, class and genus levels,

303

to further investigate the diversity of the microbial community. The three dominant

304

phyla, Proteobacteria, Bacteroidetes and Firmicutes, which have been identified in

305

anaerobic32-34 or bioelectrochemical systems,35-37 were identical but with different

306

relative abundance for all samples (Fig. 3A). At the class level, the majority of

307

sequences belonged to 10 classes, among which Bacteroidia, Clostridia,

308

Betaproteobacteria and Gammaproteobacteria were the shared predominant groups

309

(Fig. 3B). Bacteroidia, Gammaproteobacteria and Clostiridia are capable of

310

producing VFAs by utilizing soluble organics,38 which were all enhanced by FNA

311

pretreatment no matter what subsequent treatments were. Bacteroidia and Clostiridia

312

clearly increased in the AD-MEC cascading system, which may improve the

313

generation of amino acids during protein hydrolysis39 and hydrogen production,40

314

respectively.

315

To obtain more detailed information on microbial communities, the genus level

316

of different samples was examined (Fig. 3C). Macellibacteroides (class Bacteroidia),

317

which can produce lactate, HAc, HBu and iso-HBu from glucose metabolism and

318

utilize many kinds of carbohydrates,41 enriched to 6.6% in AD_FNA and further

319

accumulated to 15.6% in AD_MEC_FNA, whereas it was only 0.4% and 1.7% in

320

AD_Control and AD_MEC_Control, respectively. Petrimonas (class Bacteroidia),

321

which are closely related to the utilization of various sugars and produce HPr and

322

HAc as their primary products,42 peaked at 4.8% in AD_MEC_FNA.

323

Proteiniclasticum (class Clostridia), which cannot utilize carbohydrate but can utilize

324

soya peptone, tryptone and amino acids,43 accumulated to 4.2% in AD_FNA versus

325

0.4% in AD_Control. Acetoanaerobium (class Bacilli), which can produce HAc from

326

H2 and CO2,44 was enriched to 3.2% in the AD_MEC system. The detailed 13



327

distribution information of these four predominant anaerobic fermentation bacteria

328

(AFB) is illustrated in Fig. 4. The total relative abundance of AFB,41-49 as shown in

329

Fig. 3C, illustrated significant differences among the four samples. AFB were

330

enriched from 4.2% (AD_Control) to 13.6% (AD_FNA) and were further enhanced to

331

19.5% (AD_MEC_Control) and 34.4% (AD_MEC_FNA) after MECs cascading

332

treatment. The results demonstrated the intense solubilization and acidification of

333

FNA-pretreated WAS occurred during AD_MEC cascading system. Electrochemically

334

active bacteria (EAB), classified in a previous study,40 and which mainly attach to

335

anodes, improved the decomposition of organic matter by consuming VFAs produced

336

during pre-fermentation and converting e- and H+ (produced in the process) to H2 (Fig.

337

3C). Their distributions intuitively demonstrated that EAB emerged only in MECs

338

rather than in pre-fermentation (Fig. 4). The total relative abundances increased to 5.5%

339

of AD_MEC_Control versus 8.7% of AD_MEC_FNA, which explained the

340

phenomenon of the higher hydrogen and current generated in the latter than that in the

341

former. In addition, Ottowia, Thermomonas (class Proteobacteria) and

342

Phaeodactylibacter (class Sphingobacteria), classified as nitrate-reducing bacteria

343

(NRB), peaked at 13.7% in AD_FNA. Notably, Ottowia, which could contribute to

344

denitrification and produce N2O in the end,50 was enriched to 7.0% in AD_FNA

345

versus 3.4% in AD_Control. Its distribution was displayed in Fig. 4, which explained

346

the denitrification that occurred in the AD_FNA test.

347

Correlation between environmental variables and microbial populations

348

To further explore the plausible relationship among characteristic genera, various

349

environmental and performance measurements in WAS pre-fermentation and MECs

350

systems, including soluble carbohydrates, VFAs, NO2--N, soluble proteins, hydrogen

351

and current, we performed CCA using 16 characteristic bacteria (Fig. 5). The soluble 14


Page 14 of 29

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


352

protein and hydrogen contents as well as the current were found to be positively

353

associated with the first canonical axis (accounting for 61.9% of the variance of

354

genera distribution), whereas the other endpoints showed a negative correlation. For

355

axis 2 (explaining 31.9% of the variance), only VFAs, soluble carbohydrates and

356

NO2--N showed good positive correlations. Detailed information was shown in Table

357

S2.

358

The strength of the relationship between environmental variables and the

359

microbial community could be indicated by the length of a vector that exists in an

360

arrow-line form. As implied, soluble carbohydrates, current and hydrogen were

361

significantly relevant to the microbial community based on the length of the vector,

362

followed by VFAs and soluble proteins. Moreover, the intersection angle between

363

VFAs and soluble carbohydrates implied VFAs and soluble carbohydrates were

364

prominently correlated, and VFAs would be influenced when the soluble carbohydrate

365

content changed. Additionally, all factors were related to NO2--N. In other words, free

366

nitrite increased as VFAs and soluble carbohydrates accumulated at the beginning of

367

FNA pretreatment, whereas it decreased because of subsequent denitrification. During

368

this process, characteristic bacterial genera, such as Ottowia, Thermomonas,

369

Comamonas and Phaeodactylibacter, were enriched. As stated previously, we also

370

found that current and hydrogen had a very high positive interaction with partial

371

bacterial genera, including Olsenella, Cloacibacillus, Geobacter, Acetoanaerobium

372

and Pseudomonas, which were the common genera in AD_MEC_Control and

373

AD_MEC_FNA. Additionally, Macellibacteroides, Proteiniphilum and Petrimonas

374

were the dominant genera in AD_MEC_FNA, which further contributed to hydrogen

375

production. This is the reason why the hydrogen yield of AD_MEC_FNA was higher

376

than that of AD_MEC_Control. In this sense, the correlation between environmental 15



377

variables and characteristic genera further specified the function of the genera and

378

was consistent with the conclusion of process assessment. The CCA results confirmed

379

that the relationship between community structure and the measured variables may

380

reveal precious insight into the WAS pre-fermentation and MECs system.

381

Significance and potential implementation for practice

382

Most sludge pretreatment technologies require substantial energy input, the

383

external addition of chemical reagents and high costs, which hinder its large-scale

384

application. In this study, FNA pretreatment has been proven to be a low-cost and

385

eco-friendly technology for WAS, which can serve as an ideal approach for energy

386

recovery from a WAS pre-fermentation-MECs system. Moreover, the MECs system

387

would serve as the essential energy recovery processing unit for WWTPs due to its

388

short operation time, high energy efficiency and excellent performance.

389

Fig. 6 illustrates the enhanced energy recovery concept with the combined

390

system applied in a WWTP. In practice, FNA could be obtained in situ as a byproduct

391

from anaerobic digestion liquor during the nitritation process of wastewater treatment

392

in a WWTP.10, 51-52 The produced FNA could evaluate WAS hydrolysis and

393

acidification in anaerobic pre-fermentation and release adequate soluble substrates for

394

MECs. MECs system would accomplish complete cascade utilization of the

395

aforementioned organics by converting them to hydrogen and electric energy, which

396

is beneficial for energy recovery and cost savings. It was reported that the net benefit

397

($132600 per annum) can be achieved at 2.13 mg HNO2−N/L condition during

398

20-days digestion from the heat and energy converted by methane.10 The benefit from

399

hydrogen production was expected to be much higher than the ones from methane

400

because of its higher calorific value. Additionally, the degradation of WAS lead to

401

lower disposal and transport costs. Certainly, the potential challenges should be 16


Page 16 of 29

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


402

further assessed in WWTPs practical implementation and related analysis should be

403

focused on the life cycle, economic and ecological evaluations in whole processes for

404

ensuring sustainability.

405

Conclusion

406

This study examined the feasibility of FNA pretreatment on hydrogen recovery

407

from WAS in a pre-fermentation-MEC system by process assessment associated with

408

microbial community network analysis. FNA pretreatment was effective in enhancing

409

WAS solubilization and acidification, promoting soluble substrates cascade utilization

410

in MECs and accelerating hydrogen and energy recovery. The highest hydrogen yield

411

from FNA pretreated WAS was 1.44 mL/g VSS, which was 2.7-fold higher than that

412

obtained from un-pretreated WAS, meanwhile, the current density peaked to 11 A/m2

413

of the former while it was only 4 A/m2 of the latter. It is worth noting that FNA

414

pretreatment stimulated accumulation of functional microorganism (AFB in

415

pre-fermentation and EAB in MECs), produced abundant soluble substrates and

416

further enhanced hydrogen production. In other words, FNA pretreatment played a

417

crucial role in the construction of the WAS innate microbial community by

418

sequencing, CCA and microbial network analysis. Zero-pollution and the in situ

419

synthesis during wastewater treatment would promote the crucial implementation of

420

FNA for WAS treatment in WWTPs.

421

Supporting Information

422

Alpha diversity of four WAS samples; The eigenvalues of first two canonical

423

axes and their relationships with each environmental factor; Current generation of

424

MECs; Rarefaction curves of the four bacterial communities based on sequencing of

425

16S rRNA gene; PCoA analysis based on the unweighted UniFrac analyses.

426

Acknowledgements 17



427

This research was supported by the National Natural Science Foundation of

428

China (NSFC, Nos. 51608345, 51708386 and 21501129), by the China Postdoctoral

429

Science Foundation (Nos. 2015M570241, 2016M591416 and 2017T100170), by the

430

Open Project of Key Laboratory of Environmental Biotechnology, CAS (No.

431

kf2016004), by the Key Research and Development (R&D) Project of Shanxi

432

Province (No. 201603D321012) and the Scientific and Technological Project of

433

Shanxi Province (No. 201701D221230 and 201601D021130).

434

References

435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473

(1) Hao, X.; Wang, Q.; Cao, Y.; van Loosdrecht, M. C. Evaluating sludge minimization caused by predation and viral infection based on the extended activated sludge model No. 2d. Water Res. 2011, 45 (16), DOI 10.1016/j.watres.2011.07.013. (2) Zhao, J.; Wang, D.; Li, X.; Yang, Q.; Chen, H.; Zhong, Y.; An, H.; Zeng, G. An efficient process for wastewater treatment to mitigate free nitrous acid generation and its inhibition on biological phosphorus removal. Sci. Rep. 2015, 5, DOI 10.1038/srep08602. (3) Ni, B. J.; Yu, H. Q. Growth and storage processes in aerobic granules grown on soybean wastewater. Biotechnol. Bioeng. 2008, 100 (4), DOI 10.1002/bit.21812. (4) Guo, W. Q.; Yang, S. S.; Xiang, W. S.; Wang, X. J.; Ren, N. Q. Minimization of excess sludge production by in-situ activated sludge treatment processes--a comprehensive review. Biotechnol. Adv. 2013, 31 (8), DOI 10.1016/j.biotechadv.2013.06.003. (5) Wang, L.; Liu, W.; Kang, L.; Yang, C.; Zhou, A.; Wang, A. Enhanced biohydrogen production from waste activated sludge in combined strategy of chemical pretreatment and microbial electrolysis. Int. J. Hydrogen Energ. 2014, 39 (23), DOI 10.1016/j.ijhydene.2014.06.006. (6) Mijung Kim, S. O.; Randeep Rakwal; Chunguang Liu; Zhenya Zhang. Biohydrogen Production from Sterilized Sewage Sludge as a Substrate Using Mixed Cultures. Int. Proc. Chem., Biol. Enviorn.Eng. 2013, 51 (17), DOI 10.7763/IPCBEE. 2013. V51. 17. (7) Lu, L.; Xing, D.; Liu, B.; Ren, N. Enhanced hydrogen production from waste activated sludge by cascade utilization of organic matter in microbial electrolysis cells. Water Res. 2011, 46 (4), DOI 10.1016/j.watres.2011.11.073. (8) Appels, L.; Baeyens, J.; Degrève, J.; Dewil, R. Principles and potential of the anaerobic digestion of waste-activated sludge. Prog. Energy Combust. Sci. 2008, 34 (6), DOI 10.1016/j.pecs.2008.06.002. (9) Jiang, G.; Gutierrez, O.; Sharma, K. R.; Keller, J.; Yuan, Z. Optimization of intermittent, simultaneous dosage of nitrite and hydrochloric acid to control sulfide and methane productions in sewers. Water Res. 2011, 45 (18), DOI 10.1016/j.watres.2011.09.009. (10) Wang, Q.; Ye, L.; Jiang, G.; Jensen, P. D.; Batstone, D. J.; Yuan, Z. Free nitrous acid (FNA)-based pretreatment enhances methane production from waste activated sludge. Environ. Sci. Technol. 2013, 47 (20), DOI 10.1021/es402933b. (11) Bai, X.; Lant, P. A.; Jensen, P. D.; Astals, S.; Pratt, S. Enhanced methane production from algal digestion using free nitrous acid pre-treatment. Renew Energ. 2016, 88, DOI 10.1016/j.renene.2015.11.054. (12) Zhou, Y.; Oehmen, A.; Lim, M.; Vadivelu, V.; Ng, W. J. The role of nitrite and free nitrous acid (FNA) in wastewater treatment plants. Water Res. 2011, 45 (15), DOI 10.1016/j.watres.2011.06.025. (13) Pijuan, M.; Ye, L.; Yuan, Z. Free nitrous acid inhibition on the aerobic metabolism of poly-phosphate accumulating organisms. Water Res. 2010, 44 (20), DOI 10.1016/j.watres.2010.07.075. (14) Jiang, G.; Gutierrez, O.; Yuan, Z. The strong biocidal effect of free nitrous acid on anaerobic 18


Page 18 of 29

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530

sewer biofilms. Water Res. 2011, 45 (12), DOI 10.1016/j.watres.2011.04.026. (15) Wang, Q.; Jiang, G.; Ye, L.; Yuan, Z. Enhancing methane production from waste activated sludge using combined free nitrous acid and heat pre-treatment. Water Res. 2014, 63, DOI 10.1016/j.watres.2014.06.010. (16) Zhang, T.; Wang, Q.; Liu, Y.; Batstone, D.; Yuan, Z. Combined free nitrous acid and hydrogen peroxide pre-treatment of waste activated sludge enhances methane production via organic molecule breakdown. Sci. Rep. 2015, 5, DOI 10.1038/srep16631. (17) Li, X.; Zhao, J.; Wang, D.; Yang, Q.; Xu, Q.; Deng, Y.; Yang, W.; Zeng, G. An efficient and green pretreatment to stimulate short-chain fatty acids production from waste activated sludge anaerobic fermentation using free nitrous acid. Chemosphere. 2016, 144, DOI 10.1016/j.chemosphere.2015.08.076. (18) Guo, Z.; Liu, W.; Yang, C.; Gao, L.; Thangavel, S.; Wang, L.; He, Z.; Cai, W.; Wang, A. Computational and experimental analysis of organic degradation positively regulated by bioelectrochemistry in an anaerobic bioreactor system. Water Res. 2017, 125, DOI 10.1016/j.watres.2017.08.039. (19) Liu, W. Z.; Cai, W. W.; Guo, Z. C.; Ling, W.; Yang, C. X.; Varrone, C.; Wang, A. J. Microbial electrolysis contribution to anaerobic digestion of waste activated sludge, leading to accelerated methane production. Renew Energ. 2016, 91, DOI 10.1016/j.renene.2016.01.082. (20) Zhou, A.; Zhang, J.; Cai, W.; Sun, R.; Wang, G.; Liu, W.; Wang, A.; Yue, X. Comparison of chemosynthetic and biological surfactants on accelerating hydrogen production from waste activated sludge in a short-cut fermentation-bioelectrochemical system. Int. J. Hydrogen Energy. 2017, 42 (14), DOI 10.1016/j.ijhydene.2016.02.075. (21) A. C. Anthonisen; R. C. L.; T. B. S. Prakasam; E. G. Srinath. Inhibition of Nitrification by Ammonia and Nitrous Acid. Water Pollut. Control. 1976, 48 (5), DOI 10.2307/25038971. (22) Zhou, A.; Zhang, J.; Cai, W.; Sun, R.; Wang, G.; Liu, W.; Wang, A.; Yue, X. Comparison of chemosynthetic and biological surfactants on accelerating hydrogen production from waste activated sludge in a short-cut fermentation-bioelectrochemical system. Int. J. Hydrogen Energy. 2016, 42 (14), DOI 10.1016/j.ijhydene.2016.02.075. (23) Yang, C.; Liu, W.; He, Z.; Thangavel, S.; Wang, L.; Zhou, A.; Wang, A. Freezing/thawing pretreatment coupled with biological process of thermophilic Geobacillus sp. G1: Acceleration on waste activated sludge hydrolysis and acidification. Bioresour. Technol. 2015, 175, DOI 10.1016/j.biortech.2014.10.154. (24) Lu, L.; Xing, D.; Ren, N. Pyrosequencing reveals highly diverse microbial communities in microbial electrolysis cells involved in enhanced H2 production from waste activated sludge. Water Res. 2012, 46 (7), DOI 10.1016/j.watres.2012.02.005. (25) Zhou, A.; Zhang, J.; Varrone, C.; Wen, K.; Wang, G.; Liu, W.; Wang, A.; Yue, X. Process assessment associated to microbial community response provides insight on possible mechanism of waste activated sludge digestion under typical chemical pretreatments. Energy. 2017, 137 (15), DOI 10.1016/j.energy.2017.02.166. (26) Zhou, A.; Zhang, J.; Wen, K.; Liu, Z.; Wang, G.; Liu, W.; Wang, A.; Yue, X. What could the entire cornstover contribute to the enhancement of waste activated sludge acidification? Performance assessment and microbial community analysis. Biotechnol. Biofuels. 2016, 9, DOI 10.1186/s13068-016-0659-y. (27) Cheng, S.; Logan, B. E. Sustainable and efficient biohydrogen production via electrohydrogenesis. Proc. Natl. Acad. Sci. U. S. A.. 2007, 104 (47), DOI 10.1073/pnas.0706379104. (28) Liu, W.; Huang, S.; Zhou, A.; Zhou, G.; Ren, N.; Wang, A.; Zhuang, G. Hydrogen generation in microbial electrolysis cell feeding with fermentation liquid of waste activated sludge. Int. J. Hydrogen Energy. 2012, 37 (18), DOI 10.1016/j.ijhydene.2012.04.090. (29) Wang, B.; Wang, S.; Li, B.; Peng, C.; Peng, Y. Integrating waste activated sludge (WAS) acidification with denitrification by adding nitrite (NO2−). Biomass Bioenergy. 2014, 67, DOI 10.1016/j.biombioe.2014.05.015 (30) Luo, J.; Feng, L.; Zhang, W.; Li, X.; Chen, H.; Wang, D.; Chen, Y. Improved production of short-chain fatty acids from waste activated sludge driven by carbohydrate addition in continuous-flow reactors: Influence of SRT and temperature. Appl. Energy. 2014, 113, DOI 10.1016/j.apenergy.2013.07.006 (31) Freguia, S.; Teh, E. H.; Boon, N.; Leung, K. M.; Keller, J.; Rabaey, K. Microbial fuel cells 19



531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587

operating on mixed fatty acids. Bioresour. Technol. 2010, 101 (4), DOI 10.1016/j.biortech.2009.09.054. (32) Zhou, A.; Liu, W.; Varrone, C.; Wang, Y.; Wang, A.; Yue, X. Evaluation of surfactants on waste activated sludge fermentation by pyrosequencing analysis. Bioresour. Technol. 2015, 192, DOI 10.1016/j.biortech.2015.06.017. (33) Guo, Z.; Zhou, A.; Yang, C.; Liang, B.; Sangeetha, T.; He, Z.; Wang, L.; Cai, W.; Wang, A.; Liu, W. Enhanced short chain fatty acids production from waste activated sludge conditioning with typical agricultural residues: carbon source composition regulates community functions. Biotechnol. Biofuels. 2015, 8, DOI 10.1186/s13068-015-0369-x. (34) Sun, R.; Zhou, A.; Jia, J.; Liang, Q.; Liu, Q.; Xing, D.; Ren, N. Characterization of methane production and microbial community shifts during waste activated sludge degradation in microbial electrolysis cells. Bioresour. Technol. 2014, 175 (6), DOI 10.1016/j.biortech.2014.10.052. (35) Bonmati, A.; Sotres, A.; Mu, Y.; Rozendal, R.; Rabaey, K. Oxalate degradation in a bioelectrochemical system: reactor performance and microbial community characterization. Bioresour. Technol. 2013, 143, DOI 10.1016/j.biortech.2013.05.116. (36) Sotres, A.; DíazﬀMarcos, J.; Guivernau, M.; Illa, J.; Magrí, A.; Bonmatí, A.; Viñas, M. Microbial community dynamics in two-chambered microbial fuel cells: effect of different ion exchange membranes. J. Chem. Technol. Biotechnol. 2015, 90 (8), DOI 10.1002/jctb.4465. (37) Cerrillo, M.; Vinas, M.; Bonmati, A. Overcoming organic and nitrogen overload in thermophilic anaerobic digestion of pig slurry by coupling a microbial electrolysis cell. Bioresour. Technol. 2016, 216, DOI 10.1016/j.biortech.2016.05.085. (38) Kato, S.; Haruta, S.; Cui, Z. J.; Ishii, M.; Yokota, A.; Igarashi, Y. Clostridium straminisolvens sp. nov., a moderately thermophilic, aerotolerant and cellulolytic bacterium isolated from a cellulose-degrading bacterial community. Int. J. Syst. Evol. Microbiol. 2004, 54 (Pt 6), DOI 10.1099/ijs.0.63148-0. (39) Ariesyady, H. D.; Ito, T.; Okabe, S. Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge digester. Water Res. 2007, 41 (7), DOI 10.1016/j.watres.2006.12.036. (40) Zhang, Y.-C.; Jiang, Z.-H.; Liu, Y. Application of Electrochemically Active Bacteria as Anodic Biocatalyst in Microbial Fuel Cells. Chinese. J. Anal. Chem. 2015, 43 (1), DOI 10.1016/S1872-2040(15)60800-3. (41) Jabari, L.; Gannoun, H.; Cayol, J. L.; Hedi, A.; Sakamoto, M.; Falsen, E.; Ohkuma, M.; Hamdi, M.; Fauque, G.; Ollivier, B.; Fardeau, M. L. Macellibacteroides fermentans gen. nov., sp. nov., a member of the family Porphyromonadaceae isolated from an upflow anaerobic filter treating abattoir wastewaters. Int. J. Syst. Evol. Microbiol. 2012, 62 (Pt 10), DOI 10.1099/ijs.0.032508-0. (42) Grabowski, A.; Tindall, B. J.; Bardin, V.; Blanchet, D.; Jeanthon, C. Petrimonas sulfuriphila gen. nov., sp. nov., a mesophilic fermentative bacterium isolated from a biodegraded oil reservoir. Int. J. Syst. Evol. Microbiol. 2005, 55 (3), DOI 10.1099/ijs.0.63426-0. (43) Zhang, K. G.; Song, L.; Dong, X. Z. Proteiniclasticum ruminis gen. nov., sp. nov., a strictly anaerobic proteolytic bacterium isolated from yak rumen. Int. J. Syst. Evol. Microbiol. 2010, 60 (Pt 9), DOI 10.1099/ijs.0.011759-0. (44) Park, K. Y.; Jang, H. M.; Park, M.-R.; Lee, K.; Kim, D.; Kim, Y. M. Combination of different substrates to improve anaerobic digestion of sewage sludge in a wastewater treatment plant. Int. Biodeterior. Biodegrad. 2016, 109, DOI 10.1016/j.ibiod.2016.01.006. (45) Zhilina, T. N.; Appel, R.; Probian, C.; Brossa, E. L.; Harder, J.; Widdel, F.; Zavarzin, G. A. Alkaliflexus imshenetskii gen. nov. sp. nov., a new alkaliphilic gliding carbohydrate-fermenting bacterium with propionate formation from a soda lake. Arch. Microbiol. 2004, 182 (2), DOI 10.1007/s00203-004-0722-0. (46) Chen, S.; Dong, X. Proteiniphilum acetatigenes gen. nov., sp. nov., from a UASB reactor treating brewery wastewater. Int. J. Syst. Evol. Microbiol. 2005, 55 (Pt 6), DOI 10.1099/ijs.0.63807-0. (47) Mao, Y.; Xia, Y.; Zhang, T. Characterization of Thauera-dominated hydrogen-oxidizing autotrophic denitrifying microbial communities by using high-throughput sequencing. Bioresour. Technol. 2013, 128 (1), DOI 10.1016/j.biortech.2012.10.106. (48) Looft, T.; Levine, U. Y.; Stanton, T. B. Cloacibacillus porcorum sp. nov., a mucin-degrading bacterium from the swine intestinal tract and emended description of the genus Cloacibacillus. Int. 20


Page 20 of 29

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604

J. Syst. Evol. Microbio. 2013, 63 (6), DOI 10.1099/ijs.0.044719-0 (49) Floyd E. Dewhirst; B. J. P.; Nia Tzellas; Brittney Coleman; Julia Downes; David A. Spratt; William G. Wade. Characterization of novel human oral isolates and cloned 16S rDNA sequences that fall in the family Coriobacteriaceae: description of Olsenella gen. nov., reclassification of Lactobacillus uli as Olsenella uli comb. nov. and description of Olsenella profusa sp. nov. Int. J. Syst. Evol. Microbiol. 2001, 51, DOI 10.1099/00207713-51-5-1797. (50) Spring, S.; Jackel, U.; Wagner, M.; Kampfer, P. Ottowia thiooxydans gen. nov., sp. nov., a novel facultatively anaerobic, N2O-producing bacterium isolated from activated sludge, and transfer of Aquaspirillum gracile to Hylemonella gracilis gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol. 2004, 54 (Pt 1), DOI 10.1099/ijs.0.02727-0. (51) Wu, Q. L.; Guo, W. Q.; Bao, X.; Zheng, H. S.; Yin, R. L.; Feng, X. C.; Luo, H. C.; Ren, N. Q. Enhanced volatile fatty acid production from excess sludge by combined free nitrous acid and rhamnolipid treatment. Bioresour. Technol. 2017, 224, DOI 10.1016/j.biortech.2016.10.086. (52) Law, Y.; Ye, L.; Wang, Q.; Hu, S.; Pijuan, M.; Yuan, Z. Producing free nitrous acid – A green and renewable biocidal agent – From anaerobic digester liquor. Chem. Eng. J. 2015, 259, DOI 10.1016/j.cej.2014.07.138.

21



Figure captures

605 606

Figure 1 Changes of NO3--N and NO2--N concentrations under FNA pretreatment

607

during 24 h (A). Time-course profiles of NH4+-N concentration in the

608

whole process (B).

609

Figure 2 The cascade utilization of soluble carbohydrate (A) and protein (B) during

610

the FNA pretreatment, pre-fermentation and MEC operation, respectively.

611

Accumulation and consumption of VFAs in pre-fermentation and MEC

612

operation (C), each component content of VFAs and corresponding

613

production of methane and hydrogen yield at the end of pre-fermentation

614

and MEC operation, respectively (D and E).

615

Figure 3 Relative abundance was defined as the number of sequences per sample.

616

Taxonomic classification of sequences from the four WAS bacterial

617

communities at the phylum (A), class (B) and genus (C) levels.

618

Hierarchical clustering analysis of bacterial communities of four samples

619

(D).

620

Figure 4 Microbial network analysis of the four WAS bacterial communities; overlap

621

of the four bacterial communities based on OTU (3% distance).

622

Figure 5 Canonical correspondence analysis (CCA) between enriched genera and

623

environmental variables [soluble carbohydrates, VFAs, NO2--N, soluble

624

proteins, hydrogen and current].

625 626

Figure 6 Implement of FNA-based pre-fermentation-microbial electrolysis cells system in practice.

627

22


Page 22 of 29

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


628

Figure 1

629 630

23



631

Figure 2

632 633

24


Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


634

Figure 3

635 636

25



637

Figure 4

638 639

26


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


640

Figure 5

641 642

27



643

Figure 6

644

28


Page 28 of 29

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


645

Abstract Graphic

646 647

Synopsis

648 649 650

This study displayed the crucial role of FNA pretreatment on hydrogen and energy recovery from WAS in pre-fermentation-microbial electrolysis cells system and its potential implement in WWTP.

29


Hydrogen Recovery from Waste Activated Sludge: Role of Free

Recommend Documents