Subscriber access provided by READING UNIV
Article
Co-variation between Distribution of Microbial Communities and Biological Metabolization of Organics in Urban Sewer Systems Pengkang Jin, Xuan Shi, Guangxi Sun, Lei Yang, Yixiao Cai, and Xiaochang C. Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05121 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 4, 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.
Environmental Science & Technology 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 33
Environmental Science & Technology
1 2 3
Co-variation between Distribution of Microbial Communities and Biological Metabolization of Organics in Urban Sewer Systems
4 5
Pengkang Jin 1,*, Xuan Shi 1, Guangxi Sun 2, Lei Yang 1, Yixiao Cai 3, Xiaochang C. Wang 1
6
1. School of Environmental and Municipal Engineering, Xi’an University of Architecture
7 8 9 10 11
and Technology, Xi’an, Shaanxi Province, 710055, China 2. Sino-Danish Center for Education and Research (SDC), ,University of Chinese Academy of Sciences, Beijing, 100190, China 3. NUS Environmental Research Institute, National University of Singapore, 1 Create Way, Singapore 138602, Singapore
12 13 14 15 16 17 18 19 20 21 22
ACS Paragon Plus Environment
Environmental Science & Technology
23
ABSTRACT: Distribution characteristics and biodiversity of microbial communities were
24
studied in a 1200-m pilot sewer system. Results showed that the dominant microorganisms,
25
fermentation bacteria (FB), hydrogen-producing acetogen (HPA), sulphate-reducing bacteria
26
(SRB) and methanogenic archaea (MA) changed significantly along the sewer systems, from
27
start to the end. The distribution of the functional microorganisms could induce substrate
28
transformation and lead to the accumulation of micromolecular organics (i.e., acetic acid,
29
propionic acid and amino acid). However, substrate transformation induced by these microbes
30
was affected by environmental factors such as oxidation-reduction potential, pH and dissolved
31
oxygen. Changes in environmental conditions along the sewer resulted in the variation of
32
dominant bioreactions. FB were enriched at the beginning of the sewer, while SRB and MA
33
were found toward the end. Furthermore, based on Spearman rank correlation analysis of
34
microbial communities, environmental factors and substrates, co-variation between microbial
35
community distribution and organics metabolization along the sewer was identified. This
36
study could provide a theoretical foundation for understanding wastewater quality variation
37
during transportation from sewers to treatment plants, therefore, promoting optimization of
38
design and operation of wastewater treatment.
39 40 41 42 43 44
ACS Paragon Plus Environment
Page 2 of 33
Page 3 of 33
Environmental Science & Technology
45
INTRODUCTION
46
The urban sewer system is an important component of urban water infrastructure for sewage
47
collection and transportation. 1 As the starting point part of the entire urban sewage disposal
48
system, urban sewer systems have been studied for decades.
49
sessions of sewage quality variation were convened in Denmark, opening a new chapter in the
50
research on bioreactions in sewer system,
51
different oxygen environment effects on microbial communities, Chen et al 6 studied biofilms
52
from sediments and the inner surface of sewers by cross-cutting biofilm samples and
53
analyzing the bio-activity of microbial communities. In addition, Tanji et al
54
self-biopurification capacity of sewage by installing concrete blocks as carriers in sewer
55
systems, and after 79 days, biofilms were found on the surface of the carriers, indicating that
56
the inner environment of the sewer was suitable for biofilm growth.
57
In general, pollutants in urban sewage are easily degraded by the microbial community in the
58
sewers, and with abundant carbon, nitrogen and other nutrients, urban sewage could provide
59
substantial substrates for the growth and reproduction of microbes. Relevant study has
60
indicated that hydrolysis and acidification were the dominant bioreactions for the
61
decomposition of substrates in sewers. 8 Our previous study 9 demonstrated that larger organic
62
molecules were transformed into products with smaller molecular weights and that
63
unsaturated organics were converted into saturated organics, leading to the improvement in
64
biodegradability of the sewage. Furthermore, the transformation of VFA into acetic acid with
65
the production of CH4 and CO2 contributed to an obvious reduction in chemical oxygen
66
demand (COD) along the sewer. However, due to a lack of understanding of the
3, 4, 5
2
In 1994, the IAWQ thematic
For example, in order to understand the
ACS Paragon Plus Environment
7
studied the
Environmental Science & Technology
67
environmental factors in sewer systems, the correlation between substrate transformation and
68
microbial community distribution is unknown.
69
The urban sewer is generally an anaerobic environment. Low dissolved oxygen (DO) could
70
affect the distribution of microbial communities and then induce homologous substrate
71
transformation. Liu et al 10 investigated differences in microbial community distribution under
72
anaerobic conditions, between manholes and sewage pipes. Results showed that the dominant
73
microbial communities were significantly correlated to certain crucial anaerobic
74
environmental factors (i.e., CH4, sulfide). In addition, it is likely that changes in the content
75
and composition of organics along the sewer would lead to changes in microbial communities,
76
especially for fermentation bacteria (FB), hydrogen-producing acetogen (HPA) and
77
methanogenic Archaea (MA). Therefore, in addition to the environmental effect on sewage
78
quality, it is of particular significance to investigate the mechanism of how a variable
79
environment influences microbial communities and what the correlation is between organics
80
transformation and microbial community distribution. However, due to lack of relevant study,
81
understanding remains poor regarding the distribution characteristics and diversity of
82
microbial communities along the sewer system.
83
To investigate these mechanisms, a pilot experimental system consisting of a 1200-m-long
84
sewer was built. The variation of substrates and environment in the sewer was monitored and
85
the variation of microbial communities along the sewer was analyzed by high-throughput
86
sequencing and quantitative polymerase chain reaction (qPCR) technologies. Furthermore,
87
based on the Spearman analysis, the co-variation between microbial community distribution
88
and organic metabolization along the sewer was determined. In general, the sewage quality
ACS Paragon Plus Environment
Page 4 of 33
Page 5 of 33
Environmental Science & Technology
89
variation in sewer system resulted in the obvious differences between the design values of
90
water quality and the actual influent quality in wastewater treatment plants. Therefore, this
91
study provides a theoretical foundation for the understanding of wastewater quality variation
92
during the transportation from sewer to treatment plant, and promotes information to optimize
93
the operation of urban wastewater treatment plants.
94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110
ACS Paragon Plus Environment
Environmental Science & Technology
111
MATERIALS AND METHODS
112
Experimental setup and raw water quality
113
The pilot urban sewer system was constructed from 1200 m of 40-mm-diameter PVC pipe and
114
consisted of a total of 35 layers from the bottom to the top. Each layer was approximately 35 m
115
in length. The sewage in the pilot sewer system was gravity fed. The experiment was
116
conducted at room temperature (25 ± 2 °C) in a controlled environment where dissolved oxygen
117
(DO) was maintained at 0.3 ± 0.05 mg/L (under anaerobic conditions), which was similar to
118
that of actual sewer networks in China. During system operation, the flow rate was controlled
119
at 0.6 m/s. To avoid sedimentation in the pipe, the pipe slope and depth ratio of sewage was
120
adjusted to 5‰ and 0.6, respectively. Detailed information was provided by Jin et al. 9 Every
121
2-3 days during the pilot test, raw water was taken from a real sewer system in Xi’an, and the
122
raw water quality (COD: 358±17 mg/L; BOD5: 260±10 mg/L; TN: 45±3 mg/L; NH3-N: 38±7
123
mg/L; TP: 7.9±0.6 mg/L; pH: 7.3±0.2) was analysed. The data obtained from approximate
124
120 samples were averaged.
125
Sampling method
126
For wastewater quality sampling and analysis, nine selected sampling points were located at 0,
127
30, 100, 200, 400, 600, 800, 1000 and 1200 m from the inlet in the sewer system. Five
128
replicates at each location were taken. For each sampling point, water quality analysis
129
included COD (chemical oxygen demand), BOD5 (biochemical oxygen demand), NH3-N
130
(ammonia nitrogen), TN (total nitrogen), and TP (total phosphorous) among others.
131
For biofilm sampling, two slipknots, one at each end of the organic glass pipe in the sewer
132
system were untied. Then, adhered biofilm was peeled off with sterile cotton swabs and
ACS Paragon Plus Environment
Page 6 of 33
Page 7 of 33
Environmental Science & Technology
133
carefully placed on disposable culture dishes. The samples were fully covered by dry ice
134
during rapid transport to laboratory, and then stored at -40 °C. Due to the destructive nature of
135
the sampling, the biofilm was only sampled for once.
136
Analysis of methanol and ethanol
137
The concentration of methanol and ethanol were measured by a gas chromatograph (GC-2014
138
Shimadzu, Japan) equipped with a flame ionization detector (FID), CB-5 capillary column
139
and AOC-5000 automatic headspace sampler. The oven was maintained at 50 °C for 5 min;
140
heated to 100 °C at a rate of 5 °C/min and held for 2 min; and then heated to 200 °C at a rate
141
of 5 °C/min. Sample injection occurred at 200 °C, and the detector was operated at 280 °C.
142
Nitrogen was used as the carrier gas at a flow rate of 5.0 mL/min. The split ratio was 2:1. The
143
headspace operating conditions were: 30 min with strong shaking for sample equilibration at
144
80 °C with sampling probe held at 90 °C. The injection volume was 1.0 mL.
145
Fluorescence excitation-emission matrix analysis
146
A spectrofluorometer (FP-6500, Jasco, Japan) was used to analyze protein concentration in
147
the treated samples. During measurements, the slit widths for excitation and emission were 5
148
nm with a scanning speed of 2000 nm/min. The excitation wavelength ranged from 220 to
149
480 nm with 2 nm intervals, and the emission wavelength ranged from 280 to 570 nm with 5
150
nm intervals. The scanning of distilled water was used to eliminate water Raman scattering.
151
Molecular weight distribution analysis of organic matter
152
LC-2010AHF high-performance liquid chromatography (Shimadzu, Japan) with a Zenix
153
SEC-100 7.8×300 mm gel column was used to determine the molecular weight. The
154
wavelength of the ultraviolet detector was 254 nm. The mobile phase was a phosphate buffer
ACS Paragon Plus Environment
Environmental Science & Technology
155
solution with a pH of 7.0. The injection volume was 20 µL. The rate was set at 0.8 mL/min.
156
Polystyrene sulfonic acid sodium salt was used as a standard.
157
Analysis of pH, DO and ORP
158
The pH, DO and ORP of the sewage were measured by a HQ30d meter (HACH. USA).
159
The pH, DO and ORP of the biofilm were analyzed by microelectrodes (Unisense Denmark).
160
The tip diameters of the DO, pH and ORP microelectrodes were 10 µm. During measurement,
161
the ultrapure water, bubbled by N2, was used as the base fluid. Nylon nets immobilized the
162
biofilm. The microelectrodes were controlled to penetrate the biofilm to analyze the
163
micro-environment inside.
164
Analysis of VFAs, CH4, formic acid and other wastewater parameters
165
VFAs and CH4 were measured by gas chromatography and formic acid was measured by
166
high-performance liquid chromatography. 9 The measurements of COD, NH3-N, NO3-TN and
167
TP were processed according to the standard methods (State Environmental Protection
168
Administration of China, 2002). Carbohydrates were measured by the anthrone method. 12
169
Protein was measured by Lowry method. 11 All measurements were performed for 3 times for
170
each sample.
171
High-throughput sequencing
172
Illumina Miseq sequencing and 454 pyrosequencing were performed to analyze the
173
distribution of bacteria and Archaea in this study.
174
by Shanghai Majorbio Bio-pharm Biotechnology Co. Ltd., Shanghai, China. The PCR
175
reactions were performed three times for each sample and three parallel samples were
176
sequenced in this study. In addition, the Spearman analysis was used in this study and the
13, 14, 15
Those sequencing were conducted
ACS Paragon Plus Environment
Page 8 of 33
Page 9 of 33
Environmental Science & Technology
177
correlation of microbes and environment (p-value) are represented by gradient colours from
178
dark blue to dark red, and the significant positive or negative correlation in this analysis are
179
represented by stars (inside the gridding). The NCBI accession number is SRP118971
180
Quantitative polymerase chain reaction
181
Quantitative polymerase chain reaction (qPCR) was performed using an Applied Biosystems
182
7500 qPCR system (Applied Biosystems, Forster City, USA). To target the fermentation
183
bacteria (FB), hydA-1290F (GGTGGAGTTATGGAAGCWGCHHT) and hydA-1538R
184
(CATCCACCWGGRCAHGCCAT) were used as the forward and reverse primer respectively.
185
16
186
(ACGCAGACTCATCCCCGTG),
187
(ACTGGTNTTCCTCCTGATTTGTA),
188
(ACGCAGGCCCATCCCCGAA) were used. 17 To target the sulfate reducing bacteria (SRB),
189
dsrA-290F
190
(GTGGMGCCGTGCATGTT) were used as the forward and reverse primer respectively. 16 To
191
target the denitrifying bacteria (DNB), nirK 876 (ATYGGCGGVCAYGGCGA) and nirK
192
1040 (GCCTCGATCAGRTTRTGGTT) were used as the forward and reverse primer
193
respectively.
194
(TTCTATGGTTACGACTTVCAGGACCARTGYGG)
195
(TTCATTGCRTAGTTWGGRTAGTT) were used as the forward and reverse primer
196
respectively. 16 The analysis of blind samples was repeated 3 times. The 20 µL qPCR reaction
197
mixture was prepared in triplicate using 6.8 µL of PCR-grade water, 10 µL TaKaRa SYBR®
198
Premix Ex TaqTM (TaKaRa BIO INC. Japan), 0.4 µL of each primer, 0.4 µL of 50 × ROX
To target hydrogen-producing acetogen (HPA), probes of S-S-S.wol-0223-a-R-19 S-F-Synm-0700-a-R-23 S-S-MPOB-0222-a-R-19
(CGGCGTTGCGCATTTYCAYACVVT)
18
To
target
the
methanogenic
and
archaea
ACS Paragon Plus Environment
and
(MA),
RH3-dsr-R'
mcrA-1430F mcrA-1530R
Environmental Science & Technology
199
reference dye, 20 ng of template DNA. The thermal cycler protocol was as follows: initial
200
step for 10 s at 95°C followed by 40 cycles of 95 °C for 5 s, then maintained 56 °C for 10 s,
201
finally maintained 72 °C for 27 s. The qPCR was tested for three times. The qPCR was
202
conducted by the fluorochrome detection in PCR amplification production which differed
203
with different sequencing methods (high throughput and high sequence depth), therefore,
204
some results might not be identical.
205
Functional microbial community classification standard
206
To investigate bioreactions occurring in sewer system, the microbial communities (determined
207
by sequencing results) were classified as fermentation bacteria (FB), hydrogen-producing
208
acetogen (HPA), sulfate reducing bacteria (SRB), denitrifying bacteria (DNB), and other
209
bacteria (OB). According to the three steps theory of anaerobic fermentation, 19 the microbial
210
communities participating in the first step were represented by hydrolytic and fermentative
211
communities. In this step, the microbial communities decomposed the macromolecular
212
organic matters into micromolecular compounds (such as volatile fatty acids, alcohol among
213
others), therefore these kinds of microbial communities were classified as fermentation
214
bacteria (FB). 20, 21 In the second step, hydrogen-producing and acetate-producing bacteria
215
would consume the substrates produced in the first step and produce hydrogen and acetate,
216
which could be available to methanogens for methane formation. Therefore, this type of
217
bacteria was classified as hydrogen-producing acetogen (HPA). 22, 23 Sulfate-reducing bacteria
218
(SRB) obtain energy by oxidizing macromolecular organic compounds or molecular hydrogen
219
(H2) while reducing sulfate (SO42−) to hydrogen sulfide (H2S). 24 Denitrifying bacteria (DNB)
220
are capable of performing denitrification as part of the nitrogen cycle. This kind of bacteria
ACS Paragon Plus Environment
Page 10 of 33
Page 11 of 33
Environmental Science & Technology
221
metabolizes nitrogenous compounds using the enzyme nitrate reductase, turning nitrogen
222
oxides back to nitrogen gas or nitrous oxide.
223
bacteria were classified as OB.
25, 26
Except for FB, HPA, DNB and SRB, other
224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242
ACS Paragon Plus Environment
Environmental Science & Technology
Page 12 of 33
243
RESULTS AND DISCUSSION
244
Analysis of the biodiversity and wastewater quality in the sewer system
245
The Shannon index decreased from the start to the rear along the sewer system. The Shannon
246
index was 4.53-4.79 from 30 to 600 m along the sewer system; however, it decreased to 4
247
toward the end of the sewer from 800-1200 m. The reduction of the Shannon index showed
248
that the biodiversity changed along the sewer. 30m
Firmicutes 15.61%
Bacteroidetes 28.91%
Proteobacteria 50.94%
200m Bacteroidetes 25.54%
Firmicutes .17.56%
Proteobacteria 45.37%
600m
Firmicutes 18.95%
Bacteroidetes 27.71% Proteobacteria 33.09%
Bacteroidetes 10.06%
1000m
Proteobacteria 71.74%
249
Firmicutes 1.15%
100m
Proteobacteria Bacteroidetes Bacteroidetes 25.38% Firmicutes Chlorobi Chloroflexi Spirochaetae Candidate_division_TM7 Acidobacteria Bacteria_unclassified Proteobacteria Fusobacteria 44.11% Verrucomicrobia Cyanobacteria Synergistetes Actinobacteria Candidate_division_WS6 Others Proteobacteria Bacteroidetes Firmicutes Chlorobi Chloroflexi Spirochaetae Candidate_division_TM7 Acidobacteria Bacteria_unclassified Fusobacteria Verrucomicrobia Cyanobacteria Synergistetes Actinobacteria Candidate_division_WS6 Others Proteobacteria Bacteroidetes Firmicutes Chlorobi Chloroflexi Spirochaetae Candidate_division_TM7 Acidobacteria Bacteria_unclassified Fusobacteria Verrucomicrobia Cyanobacteria Synergistetes Actinobacteria Candidate_division_WS6 Others Proteobacteria Bacteroidetes Firmicutes Chlorobi Chloroflexi Spirochaetae Candidate_division_TM7 Acidobacteria Bacteria_unclassified Fusobacteria Verrucomicrobia Cyanobacteria Synergistetes Actinobacteria Candidate_division_WS6 Others
400m
Firmicutes 17.40%
Firmicutes 12.18%
Bacteroidetes 25.16%
Proteobacteria 44.82%
Bacteroidetes 9.89%
800m
Firmicutes 2.44%
Proteobacteria 83.20%
Bacteroidetes 9.94%
1200m
Proteobacteria 72.01%
ACS Paragon Plus Environment
Firmicutes 0.35%
Proteobacteria Bacteroidetes Firmicutes Chlorobi Chloroflexi Spirochaetae Candidate_division_TM7 Acidobacteria Bacteria_unclassified Fusobacteria Verrucomicrobia Cyanobacteria Synergistetes Actinobacteria Candidate_division_WS6 Others Proteobacteria Bacteroidetes Firmicutes Chlorobi Chloroflexi Spirochaetae Candidate_division_TM7 Acidobacteria Bacteria_unclassified Fusobacteria Verrucomicrobia Cyanobacteria Synergistetes Actinobacteria Candidate_division_WS6 Others Proteobacteria Bacteroidetes Firmicutes Chlorobi Chloroflexi Spirochaetae Candidate_division_TM7 Acidobacteria Bacteria_unclassified Fusobacteria Verrucomicrobia Cyanobacteria Synergistetes Actinobacteria Candidate_division_WS6 Others Proteobacteria Bacteroidetes Firmicutes Chlorobi Chloroflexi Spirochaetae Candidate_division_TM7 Acidobacteria Bacteria_unclassified Fusobacteria Verrucomicrobia Cyanobacteria Synergistetes Actinobacteria Candidate_division_WS6 Others
Page 13 of 33
Environmental Science & Technology
250
Fig. 1 Relative abundance of microbial communities at the phylum level along the sewer
251
(Others in each plot represented the unidentified bacteria)
252
As shown in Fig. 1, the relative abundance of Proteobacteria was 30.08-50.94% within the
253
beginning 30-600 m of the sewer, however, from 600 to 800 m, Proteobacteria became
254
dominant with a relative abundance of 71.7-83.2%. In contrast, in the first 600 m of the sewer,
255
the relative abundance of Bacteroidetes was 25.2-28.9%, and, rapidly decreased to
256
approximately 10% at 800-1200 m. Moreover, within 30-600 m, the relative abundance of
257
Fimicutes was 12.2-18.9%, however, it dramatically decreased to less than 2.5% at 800-1200
258
m.
259
In fact, Firmicutes, Bacteroidetes and Proteobacteria contain the fermentative genera which
260
contribute to the fermentation processes in the anaerobic digestion system. 27 Moreover, the
261
environmental conditions of the sewer system are dark and anaerobic, similar to anaerobic
262
digestion systems, indicating that fermentation might occur in a sewer system. In general,
263
hydrolysis and acidification are components of fermentation process. During hydrolysis,
264
refractory organic matter is decomposed into degradable compounds, and macromolecular
265
organic matter is decomposed into small molecules. On the other hand, acidification produces
266
acid and gases. Kang et al
267
and acidification during sludge fermentation, suggesting that the competitive capacity of
268
Proteobacteria could be stronger at the end of the sewer, where acidification might be the
269
primary process (accumulation of small-molecule organic matters as shown in supporting Fig.
270
3). It has been reported that Bacteroidetes and Firmicutes could hydrolyze protein and
271
carbohydrates.
29, 30
28
found that Proteobacteria was the major phylum in hydrolysis
As the content of macromolecular organic compounds (molecular
ACS Paragon Plus Environment
Environmental Science & Technology
272
weight > 10000 Dalton and within 650-10000 Dalton) decreased along the sewer (Supporting
273
Fig. 3), and the relative abundance of Bacteroidetes and Firmicutes similarly decreased, it can
274
be inferred that the local environment became disadvantageous for Bacteroidetes and
275
Firmicutes due to lack of metabolizable substrates.
276
To study the effect of sewer length on the microbial community (bacteria and Archaea)
277
distribution, the beta-diversity as principal component analysis (PCA) plots were produced
278
(Supporting Fig. 1), and the microbial communities in each biofilm sample were represented
279
as colored points. The results showed that significant separation of bacteria occurred in the
280
samples from 0-200 m, 400-600 m and 800-1200 m (Supporting Fig. 1(a)). It indicated that
281
due to the variation of environmental factors along the sewer, the diversity (Beta) of bacteria
282
was affected along the range of 400-600 m (diversity changed significantly in the beginning
283
of the sewer). The separation groups of Archaea were found in the samples from 0-400 m,
284
600-800 m and 1000-1200 m (Supporting Fig. 1(b)). The results indicated that the diversity
285
(Beta) of Archaea could be affected in the range of 600-800 m of sewer. The PCA analysis
286
was in accordance with the results shown in Fig. 1. The analysis of microbial community
287
diversity and structures at the phylum level along the length of sewer could provide a
288
profound understanding on microenvironments and the investigation of the diverse
289
metabolism occurring in sewer system.
290
Distribution characteristics of functional microbial communities in the sewer system
ACS Paragon Plus Environment
Page 14 of 33
Page 15 of 33
Environmental Science & Technology
Concentration of 16S rRNA gene copy (copied/ml)
1.00E+13
1.00E+11
1.00E+09
30m
100m
200m
400m
600m
800m
1000m
1200m
1.00E+07
1.00E+05
1.00E+03 FB
HPA
SRB
DNB
Funtional microbial communities
291
(a)
292 6.00E+03
1.60E+04
DNB DNB SRB
1.20E+04 OHB OB
DNB DNB SRB HPA
32.5% 31.6% 31.6%
Reads
Relative Abundance
4.00E+03
Reads
38.3%
5.00E+03
OHB OB
DNB DNB
OHB OB
OHB OB
OB OHB
OHB OB
OHB OB
DNB 4.00E+03
SRB
SRB
HPA
HPA
0.00E+00 30
3.00E+03
DNB DNB
29.7%
21.1% 2.00E+03 17.4%
OB OHB
8.00E+03
400
600
DNB DNB HPA
FB FB
200
800
1000
1200
Length of the sewer system (m)
SRB SRB
HPA
FB
100
DNB DNB
SRB
FB
HPA FB
FB
1.00E+03
DNB
5.8%
SRB FB
HPA FB
0.00E+00 30
OB OHB OHB
293 294
` Desulfonema Enterobacter Lactococcus
100
200
Thauera Desulfovibrio Paludibacter Tolumonas
400
600
800
1000
Length of the sewer system (m) Alicycliphilus Dechloromonas Anaerolinea Veillonella Flavobacterium Aeromonas Lactobacillus Cloacibacterium
1200
Desulfobulbus Anaeroarcus Raoultella Trichococcus
(b)
295
Fig. 2 Variation of fermentation bacteria (FB), hydrogen-producing acetogen (HPA), sulfate
296
reducing bacteria (SRB), denitrifying bacteria (DNB), and other bacteria (OB) along the
297
sewer (a) 16S rRNA gene copy concentration of functional microbial communities (detected
298
by qPCR); (b) relative abundance of functional microbial communities (detected by
ACS Paragon Plus Environment
Environmental Science & Technology
299
sequencing ). (The data and detailed classification of each functional microbial community
300
was shown in Supporting Table 1 and Table 2)
301
Because pyrosequencing only provided information on the relative abundance of microbial
302
communities rather than the absolute quantity variation, quantitative polymerase chain
303
reaction (qPCR) and pyrosequencing were used together to investigate the distribution
304
characteristics of functional microbial communities along the sewer. The variation of the 16S
305
rRNA gene copy concentration and the relative abundance at the genus level are shown in Fig.
306
2. It was observed that the functional microbes in the sewer were mainly comprised of
307
fermentation bacteria (FB), hydrogen-producing acetogen (HPA), sulfate reducing bacteria
308
(SRB) and denitrifying bacteria (DNB).
309
As shown in Fig. 2 (a), the 16S rRNA gene copy concentration of FB continuously increased
310
along the 0-600 m range of the sewer and reached the maximum at 600 m (2.39E+12
311
copied/ml). It then began to decrease significantly from 600 m to 1200 m. These results
312
indicated that: within 600-1200 m of the sewer, some communities of FB declined in
313
population, and the microbial community structures and biometabolism processes might be
314
altered in this region. To understand this phenomenon, pyrosequencing was utilized to reveal
315
the variation of microbial community structures and then infer the bioreactions that occurred
316
in the sewer system. In the first 30 m of the sewer system, Trichococcus and Cloacibacterium
317
were the dominant genera. As the distance increased, Cloacibacterium was gradually
318
eliminated. Previous studies indicated that Trichococcus could adapt to the environment
319
where hydrolysis substrates, (especially macromolecular organic compounds) were rich. 31
320
Therefore, along the sewer from 0 to 400 m, due to the high content and complicated
ACS Paragon Plus Environment
Page 16 of 33
Page 17 of 33
Environmental Science & Technology
321
composition of macromolecular organic compounds (Supporting Fig. 3), the environment
322
might be more suitable for the growth of Trichococcus (which displayed high relative
323
abundance in this region). From 600 to 800 m, the relative abundance of Trichococcus
324
decreased dramatically, indicating that Trichococcus might no longer adapt to the changing
325
environment. This result was highly consistent with the qPCR data which indicated that the
326
16S rRNA gene copy concentration of FB decreased significantly after 600 m. Moreover, it
327
also indicated that the change of FB concentration was mainly due to the elimination of
328
Trichococcus. However, the relative abundance of Flavobacterium significantly increased,
329
making it the dominant genus in this region. This might be resulted from the accumulation of
330
micromolecule compounds as substrates for its reproduction (Supporting Fig. 3). After 800 m,
331
as the substrates were gradually consumed, the relative abundance of Flavobacterium and the
332
16S rRNA gene copy concentration of FB continuously decreased along the sewer, indicating
333
that the substrates were more readily utilized by other genera at the end of the sewer. Hence,
334
these results indicated that the distribution characteristics of FB featured the dominant genus
335
changing from Trichococcus to Flavobacterium within 600-800 m of the sewer system.
336
HPA mainly consumed hydrolysate for final acidification. The 16S rRNA gene copy
337
concentration of HPA remained low in the beginning of sewer, with no accumulation of
338
(hydrolytic products), and then it increased dramatically after 100 m as hydrolysis improved
339
along the sewer (Fig. 2 (a)). Moreover, the distribution characteristics of HPA changed in the
340
sewer, i.e., the dominant HPA genus changed from Veillonella to Anaerolinea within the
341
600-800 m region. Therefore, the variation of the HPA structure also caused the decline of
342
HPA concentration after 600 m (Fig. 2 (a)). Previous studies have showed that Veillonella and
ACS Paragon Plus Environment
Environmental Science & Technology
Page 18 of 33
32, 33
343
Anaerolinea prefer to utilize lactic acid and small-molecule fatty acids respectively.
344
Therefore, the accumulation of lactic acid at the beginning of the sewer (supporting Fig. 5)
345
might be the reason for the high relative abundance of Veillonella. When the content of lactic
346
acid decreased near the end of the sewer, the reproduction of Veillonella was limited while
347
Anaerolinea became dominant perhaps due to the stable concentration of small-molecule fatty
348
acids (VFA).
349
As shown in Fig. 2(b), the relative abundance of Desulfovibrio was higher than that of other
350
SRB genera at 30 m. Previous reports indicated that Desulfovibrio preferred to utilize
351
methanol and lactic acid as carbon source. 34 From 100 to 400 m, the relative abundance of
352
Desulfovibrio decreased gradually, while Desulfonema became the dominant genus, which
353
might be due to the accumulation of isobutyric acid, the preferable carbon source for
354
Desulfonema, in this region (supporting Fig. 5). 35 However, due to the low content of SO42-,
355
the relative abundance of all SRB genera decreased beyond 600 m in the sewer, suggesting
356
that the sulfate reduction process had gradually declined. This was also verified by the qPCR
357
data that showed that the 16S rRNA gene copy concentration of SRB decreased after 600 m.
358
These results suggest that the distribution characteristics of SRB changed along the sewer
359
with the dominant genus changing from Desulfovibrio to Desulfonema.
360
Due to the anaerobic environment in sewer systems, nitrification was inhibited. Therefore, the
361
content of NO3- was extremely low (Supporting Fig. 4). Previous study has shown that
362
Dechloromonas and Alicycliphilus can reduce NO3-, 36 however, due to the low content of
363
NO3- in the sewer, the metabolism of these two dominant DNB genera might be weakened.
364
Moreover, the total concentration of DNB was also low in the sewer.
ACS Paragon Plus Environment
Page 19 of 33
Environmental Science & Technology
365
Except for microbial genera discussed above, all other genera could be regarded as a whole
366
microbial community, namely, other bacteria (OB). The relative abundance of OB varied from
367
60% to 95% along the sewer system, suggesting that the competition capacity of functional
368
bacteria mentioned above were gradually weaker than OB, and the OB were the most
369
dominant microbial communities at the end of sewer. Concentration of 16S rRNA gene copy (copied/ml)
1.00E+11
1.00E+09
1.00E+07
1.00E+05
1.00E+03
100m
200m
400m
600m
800m
1000m
1200m
1000
1200
Lenth of the sewer system (m)
370 1.20E+04
100 %
Reads Relative Abundance
1.00E+04
75 %
8.00E+03
6.00E+03 50 % 4.00E+03
25 % 2.00E+03
0.00E+00 0% 100
371 372
200
400 600 800 Length of the sewer system (m)
Others
Methanosaeta
Archaea
Methanobacterium
Methanospirillum
Methanosarcinaceae
Methanobacteriaceae
Euryarchaeota
Methanosarcina
Fig. 3 Variation of methanogenic Archaea (MA) along the sewer (a) 16S rRNA gene copy
ACS Paragon Plus Environment
Environmental Science & Technology
373
concentration of MA (detected by qPCR); (b) relative abundance of MA ((detected by
374
sequencing)
375
The production of methane in the sewer system indicated the existence of methanogenic
376
Archaea (MA). Most likely due to lack of metabolizable substrates available to MA at the
377
beginning of the sewer, MA was not found at the 30 m location (verified by both qPCR and
378
pyrosequencing detection as shown in Fig. 3). The 16S rRNA gene copy concentration of MA
379
increased from 100 m to 1000 m of the sewer, which indicated that methanogenesis could be
380
gradually enhanced along the sewer. To acquire the specific information on methanogenesis,
381
the MA taxa were identified. The dominant MA genera were Methanosarcina, Euryarchaeota
382
and Methanobacteriaceae, among which a transition from Methanosarcina to Euryarchaeota
383
was clearly observed. It was reported that Methanosarcina was able to use aceticlastic and
384
hydrogenotrophic pathways, as they are more adaptable to changes in nutritional conditions. 37
385
The relative abundance of Methanosarcina continuously increased from 100 to 800 m, and
386
exceeded that of Euryarchaeota at 200 m while the relative abundance of Euryarchaeota
387
decreased continously along the sewer. This indicates that Methanosarcina adapted to grow in
388
the environment where the substrates were rich in complex substrate conditions from 100 to
389
600 m. However, after 800 m, the relative abundance of Methanosarcina decreased while that
390
of Euryarchaeota increased along 800-1200 m. This suggested that the substrate conditions
391
from 800 to 1200 m were more suitable for the growth of Euryarchaeota. Based on the
392
analysis of the functional microbial communities distribution along the sewer discussed above,
393
the metabolism can be inferred. The metabolism occurring in sewer would be affected by
394
environmental factors, and therefore cause the substrate transformation. Therefore, the
ACS Paragon Plus Environment
Page 20 of 33
Page 21 of 33
Environmental Science & Technology
395
variation of nutrient substrate and environmental conditions along the sewer were further
396
studied
397
The variation of nutrient substrate and environmental conditions along the sewer protein
tryptophan-like 类色氨酸
类腐殖质 humic-like 类腐殖质
>10000Da
800
200
600
150
400
100
200
50
0 0
200
400 600 800 Lenth of the sewer system (m)
1000
650 Dalton) continuously declined while the small-molecule
416
organics (< 650 Dalton) rose constantly. This indicated that the macromolecular organic
417
compounds were continuously decomposed to small-molecule organics in the 800-1200 m
418
region, which might retard hydrolysis and facilitate acidification. Thus, the environment
419
might be more suitable for the growth of Flavobacterium (FB), Anaerolinea (HPA),
420
Desulfonema (SRB), and Euryarchaeota (MA) near the end of sewer due to the enhancement
421
of acidification. pH of sewage
pH of biofilm
DO of sewage
DO of biofilm
200 15
400 30
600 45
800 60
1000 75
1
7
0.8
6
0.6
pH
8
5
0.4
4
0.2
3
0 0
200
400
600
800
1000
422
Length of the sewer system (m)
423
(a)
1200
ORP of biofilm
Passing time (min)
0 1200 90
00
200 15
400 30
600 45
800 60
1000 75
1200 90
0
200
400
600
800
1000
1200
300 200 100
ORP (mV)
0
Concentration of DO (O2mg/L)
Passing time (min)
3.2
ORP of sewage
0 -100 -200 -300 -400 0
-500 Length of the sewer system (m)
(b)
424
Fig. 5 Variation of pH, DO and ORP along the sewer system (measured five times)
425
To further study the influence of environmental condition on microbial communities in the
426
sewer, pH, DO, and ORP of the sewage and biofilm were measured. As shown in Fig. 5 (a),
427
the concentration of DO in the sewage and biofilm decreased gradually along the sewer from
428
0.5 mgO2/L to 0.19 mgO2/L, and from 0.38 mgO2/L to 0.16 mgO2/L, respectively, while the
429
pH values of the sewage and biofilm both remained stable (6.0-7.0). Therefore, the sewer was
430
anaerobic. Moreover, the anaerobic conditions in both sewage and biofilm became more
ACS Paragon Plus Environment
Page 23 of 33
Environmental Science & Technology
431
pronounced along the sewer. Fig. 5 (b) presents the change of ORP in sewage and biofilm.
432
The ORP of sewage decreased from 175 mV to -70 mV along the sewer. In the first 600 m,
433
the ORP of the biofilm decreased steadily along the sewer, however, it decreased rapidly from
434
-200 to -306 mV between 600 and 800 m. The rate of decrease slowed at the end of the sewer.
435
The ORP variation in the biofilm affected biochemical reactions and therefore could influence
436
the change of substrates in the sewer. As shown in Fig. 5 (b), the ORP of biofilm ranged from
437
-100 to -200 mV between 30 and 600 m in the sewer. As reported previously, the optimal ORP
438
ranges for acidogenic fermentation and sulfate reduction are from -100 to -225 mV and from
439
-50 to -250 mV, respectively 38. Therefore, it can be inferred that acidogenic fermentation and
440
sulfate reduction mainly occurred along the 30-600 m segment the sewer system. This result
441
was verified by the distribution of acidogenic fermentation microflora (i.e., high relative
442
abundance of FB and HPA) in the beginning of sewer (Fig. 2). In contrast, despite of the
443
favorable ORP condition for SRB reproduction, the relative abundance of SRB remained low
444
probably due to the low content of SO42- (less than 50 mg/L along the sewer, Supporting Fig.
445
4). From 800 to 1000 m, the ORP of biofilm ranged from -300 to -350 mV, therefore,
446
acidogenic fermentation and sulfate reduction was reduced and the relative abundance of the
447
corresponding functional microbes was lower than that in the 0-600 m segment. Moreover,
448
the suitable ORP for denitrification ranged from 50 to -50 mV 38, therefore, along the entire
449
sewer the relative abundance of DNB genera was lower than that of other functional microbes.
450
In addition, the optimal range of ORP for the methane production is between -175 to -400 mV,
451
38
452
production. As shown in Fig. 4, from 100 to 800 m, the relative abundance of Methanosarcina
thus the ORP condition along the majority part of the sewer was beneficial to methane
ACS Paragon Plus Environment
Environmental Science & Technology
Page 24 of 33
453
was the highest among MA with ORP between -147 and -306 mV, suggesting that the ORP
454
condition was highly suitable for Methanosarcina growth. When ORP decreased to -356 mV,
455
the relative abundance of Euryarchaeota was the highest among the MA, indicating that the
456
ORP condition was more beneficial for the growth of Euryarchaeota.
457
Within 0-200 m of the sewer, fermentation products continuously increased, including lactic
458
acid, acetic acid, propionic acid and isobutyric acid, of which lactic acid and acetic acid are
459
the dominant products and contribute to the highest ratio (Supporting Fig. 5). The content of
460
lactic acid reached its maximum value of 7.89 mg/L at 200 m. In the 200-600 m segment of
461
the sewer, lactic acid and ethanol concentration was reduced while the content of propionic
462
acid and isobutyric acid increased continuously. At the 600 m location, the content of acetic
463
acid, propionic acid and isobutyric acid increased to 8.67 mg/L, 3.5 mg/L and 2.5 mg/L,
464
respectively, and it was corresponded with the analysis of molecular weight that demonstrated
465
that the macromolecular organic compounds were gradually decomposed to small-molecule
466
organic compounds along the sewer systems.
467
The
468
communities
co-variation
DO
between
pH
environmental
Ethanol
condition,
Lactic acid Formic acid Acetic acid Propionic acid
FB
SRB HPA DNB 469
substrate
Archaea
ACS Paragon Plus Environment
and
microbial
Page 25 of 33
Environmental Science & Technology
470
Fig. 6 Correlations between microbiota abundances and environment conditions (based on
471
Spearman' s correlation between the relative abundance of significantly functional microbes
472
and the environment condition in the sewer. The color and the stars of each stripe indicate
473
magnitude of correlation, with p-values superimposed on stripe (One star represents p<0.05,
474
and two stars represent p<0.01); Subject groups were divided in FB, SRB, HPA, DNB and
475
Archaea; the alpha values and water quality along the sewer are shown in supporting Table 5,
476
Table 6)
477
To assess the correlation between sewage quality variables and microbial communities on
478
bioflims in the sewer, the Spearman analysis was conducted to visualize the correlation
479
between functional microorganism and environmental factors. As shown in Fig. 6, FB
480
exhibited a significantly positive correlation (red cell with stars) with macromolecular
481
organics, especially ethanol and lactic acid, therefore the levels of DO and pH in sewer could
482
remarkably affect the metabolism of FB. These results were highly consistent with the
483
previous discussion that FB became the dominant microflora as a result of the abundant
484
macromolecular organics available as substrate in the beginning of sewer system while the
485
relative abundance of FB gradually decreased along the sewer. Nevertheless, it should be
486
noted that Flavobacterium (FB) showed positive correlation with small-molecule organics
487
(acetic acid and propionic acid), and the bacteria could survived in the sewer despite
488
variations in DO and pH. Therefore, FB could still be detected at the end of sewer even
489
though their relative abundance was quite low. HPA and SRB exhibited a positive correlation
490
with lactic acid (red cell), therefore, abundant lactic acid at the beginning of the sewer enabled
491
HPA and SRB to be more competitive and display greater relative abundance. The utilization
ACS Paragon Plus Environment
Environmental Science & Technology
492
of lactic acid as a carbon source by Veillonella (the dominant genus of HPA) and
493
Desulfovibrio (the dominant genus of SRB) led to the continuous decrease of lactic acid. Thus,
494
beyond 600 m, due to the lower content of lactic acid, the distribution characteristics of HPA
495
changed. Meanwhile, due to lack of SO42-, the relative abundance of SRB decreased within
496
800-1200 m. DNB (Alicycliphilus and Thauera) exhibited significantly negative correlation
497
with carbon availability (blue cell with stars) although Dechloromonas could use carbon for
498
denitrification. This is due to the extremely low content of NO3- was in sewer. A complex
499
substrate composition for MA was observed, which included methanol, lactic acid, formic
500
acid and propionic acid. The relative abundance of Methanosarcina was higher before 800 m.
501
However, beyond 800 m, acetic acid contributed nearly all of the substrate for MA and the
502
relative abundance of Euryarchaeota became the highest among the MA. Thus, it can be
503
inferred that Methanosarcina could adapt to the complex substrate conditions while
504
Euryarchaeota tended to prefer acetic acid as a substrate.
505
In conclusion, this study revealed the distribution characteristics of different functional
506
microbial communities (FB, HPA, SRB, DNB, MA). Due to the variation of ORP, DO and pH,
507
the dominant bioreaction changed from fermentation to methane production along the sewer.
508
These results indicated that different environmental factors could affect the distribution of
509
functional microbial community, and the change of dominant FB, HPA, SRB, DNB and MA
510
genera along the sewer could induce homologous transformation of organics. The distribution
511
of microbial communities and organic metabolism are inseparably connected along sewer
512
systems. This study could provide a theoretical foundation for an understanding of wastewater
513
quality variation along sewer transport systems. Thus, these results could help to predict the
ACS Paragon Plus Environment
Page 26 of 33
Page 27 of 33
Environmental Science & Technology
514
actual influent quality of wastewater treatment plants, and promote optimization of wastewater
515
treatment.
516
AUTHOR INFORMATION
517
Corresponding Author
518
Pengkang Jin, Phone: +86 13379217572; E-mail:
[email protected] 519
Author Contributions
520
The manuscript was written by contributions of all authors. All authors have approved the
521
final version of the manuscript.
522
Notes
523
The authors declare no competing financial interest.
524
ACKNOWLEDGEMENTS
525
This study was supported by the National Key Project of Water Pollution Control and
526
Management (No. 2012ZX07313001), Shaanxi Science & Technology Co-ordination &
527
Innovation Project (2016TZC-S-19-3), and the Program for Innovative Research Team in
528
Shaanxi Province (PIRT) (No. 2013KCT-13).
529
SUPPORTING INFORMATION AVAILABLE
530
Including the relative abundance of functional microbial community; the variation of substrate
531
concentrations; the variation of environmental factors along the sewer. This information is
532
available free of charge via the Internet at http://pubs.acs.org.
533
REFERENCES
534
(1) Mathioudakis, V.L; Aivasidis, A. Heterotrophic denitrification kinetics in a pressurized
535
sewer biofilm reactor. Desalination. 2009, 248 (1), 696-704.
ACS Paragon Plus Environment
Environmental Science & Technology
536 537
(2) Chen, G.H; Leung, D.H.W; Huang, J.C. Removal of dissolved organic carbon in sanitary gravity sewer. J. Environ. Eng. 2001, 127 (4), 295-301.
538
(3) Verbanck, M.A; Ashley, R.M; Bachoc, A. International workshop on origin, occurrence
539
and behaviour of sediments in sewer systems: Summary of conclusions. Water Res. 1994,
540
28 (1), 187-194.
541 542
(4) Ashley, R.M; Arthur, S; Coghlan, B.P. Fluid sediment in combined sewers. Water Sci Technol. 1994, 29 (1-2), 113-123.
543
(5) Okabe, S; Odagiri, M; Ito, T; Satoh, H. Succession of sulfur-oxidizing bacteria in the
544
microbial community on corroding concrete in sewer systems. Appl. Environ. Microbiol.
545
2007, 73 (3), 971-980.
546 547 548 549 550 551
(6) Chen, G.H; Leung, D.H.W; Hung, J.C. Biofilm in the sediment phase of a sanitary gravity sewer. Water Res. 2003, 37 (11), 2784-2788. (7) Tanji, Y; Sakai, R; Miyanaga, K; Unno, H. Estimation of the self-purification capacity of biofilm formed in domestic sewer pipes. Biochem. Eng. J. 2006, 31 (1), 96-101. (8) Hvitved-Jacobsen, T; Vollertsen, J; Nielsen, A.H. Sewer processes: Microbial and Chemical Process Engineering of Sewer Networks. CRC press. 2013.
552
(9) Jin, P.K; Wang, B; Jiao, D; Sun, G.X; Wang, B.B; Wang, X.C. Characterization of
553
microflora and transformation of organic matters in urban sewer system. Water Res. 2015.
554
84, 112-119.
555
(10) Liu, Y; Dong, Q; Shi, H; Distribution and population structure characteristics of
556
microorganisms in urban sewage system. Appl. Microbiol. Biotechnol. 2015, 99 (18),
557
7723-7734.
ACS Paragon Plus Environment
Page 28 of 33
Page 29 of 33
558 559 560 561
Environmental Science & Technology
(11) Dreywood, R. Qualitative test for carbohydrate material. Ind. Eng. Chem. Res. 1946, 18 (8), 499-499. (12) Lowry, O, H; Rosebrough, N, J; Farr, A, L; Randall, R, J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 1951, 193, 265-275.
562
(13) Liang, X; Fu, Y; Liu, H. Isolation and characterization of enzyme-producing bacteria of
563
the silkworm larval gut in bioregenerative life support system. Acta Astronautica. 2015,
564
116, 247-253.
565 566
(14) Horner-Devine, M, C; Lage, M; Hughes, J, B; Bohannan, B, J. A taxa–area relationship for bacteria. Nature. 2004. 432 (7018), 750-753.
567
(15) Luton, P, E; Wayne, J, M; Sharp, R, J; Riley, P, W. The mcrA gene as an alternative to
568
16S rRNA in the phylogenetic analysis of methanogen populations in landfill.
569
Microbiology. 2002, 148 (11), 3521-3530.
570
(16) Pereyra, L, P; Hiibel, S, R; Riquelme, M, P; Reardon, K, F; Pruden, A. Detection and
571
quantification
572
sulfate-reducing bacteria and methanogenic archaea. Appl Environ Microbiol. 2010, 76
573
(7), 2192-2202.
574 575
of
functional
genes
of
cellulose-degrading,
fermentative,
and
(17) Xu, K, W. A microbial ecological study on the mechanism of acetate accumulation during anaerobic incubation of sewage sludge. Jiangnan university. 2010.
576
(18) Kim, Y, M; Lee, D, S; Park, C; Park, D; Park, J, M.. Effects of free cyanide on microbial
577
communities and biological carbon and nitrogen removal performance in the industrial
578
activated sludge process. Water Res. 2011, 45 (3), 1267-1279.
579
(19) Bryant, M, P. Microbial methane production—theoretical aspects. J Anim Sci. 1979, 48
ACS Paragon Plus Environment
Environmental Science & Technology
580
Page 30 of 33
(1), 193-201.
581
(20) Buchanan, R.E; Gibbons, N.E; Bergeys Manual of Determinative Microbiology. 1974
582
(21) Ishii, S.I; Suzuki, S; Norden-Krichmar, T.M; Phan, T; Wanger, G; Nealson, K.H;
583
Bretschger, O; Microbial population and functional dynamics associated with surface
584
potential and carbon metabolism. ISME J, 2014. 8 (5), 963-978.
585 586
(22) Rogosa, M. The Genus Veillonella I. General Cultural, Ecological, and Biochemical Considerations. J. Bacteriol. 1964. 87 (1), 162-170.
587
(23) Yang, Q; Xiong, P; Ding, P; Chu, L; Wang, J. Treatment of petrochemical wastewater by
588
microaerobic hydrolysis and anoxic/oxic processes and analysis of bacterial diversity.
589
Bioresour. Technol. 2015. 196, 169-175.
590 591 592
(24) Muyzer, G. and Stams, A, J. The ecology and biotechnology of sulphate-reducing bacteria. Nat Rev Microbiol. 2008. 6, 441–454. (25) Achenbach,
L.A;
Michaelidou,
U; Bruce,
R.A;
Fryman,
J;
Coates,
J.D.
593
Dechloromonasagitata gen. nov., sp. nov.andDechlorosomasuillum gen. nov., sp. nov.,
594
two novel environmentally dominant (per) chlorate-reducing bacteria and their
595
phylogenetic position. Int J Syst Evol Microbiol. 2001. 51 (2), 527-533.
596
(26) Han, X; Wang, Z; Ma, J; Zhu, C; Li, Y; Wu, Z. Membrane bioreactors fed with different
597
COD/N ratio wastewater: impacts on microbial community, microbial products, and
598
membrane fouling. Environ. Sci. Pollut. Res. 2015. 22 (15), 11436-11445.
599 600 601
(27) Nelson, M, C; Morrison, M; Yu, Z. A meta-analysis of the microbial diversity observed in anaerobic digesters. Bioresour. Technol. 2011. 102 (4), 3730-3739. (28) Kang, X, R; Zhang, G, M; Chen, L; Dong, W, Y; Tian, W, D. Effect of initial pH
ACS Paragon Plus Environment
Page 31 of 33
Environmental Science & Technology
602
adjustment on hydrolysis and acidification of sludge by ultrasonic pretreatment. Ind. Eng.
603
Chem. Res. 2011. 50 (22), 12372–12378.
604
(29) Ueki, A; Akasaka, H; Suzuki, D; Ueki, K. Paludibacter propionicigenes gen. nov., sp.
605
nov., a novel strictly anaerobic, Gram-negative, propionate-producing bacterium isolated
606
from plant residue in irrigated rice-field soil in Japan. Int J Syst Evol Microbiol. 2006. 56
607
(1), 39–44.
608
(30) Leven, L; Eriksson, A, R; Schnürer, A. Effect of process temperature on bacterial and
609
archaeal communities in two methanogenic bioreactors treating organic household waste.
610
FEMS Microbiol. Ecol. 2007. 59 (3), 683–693.
611
(31) Buchanan, R, E; Gibbons, N, E. Bergeys Manual of Determinative Microbiology. 1974.
612
(32) Rogosa, M. THE GENUS VEILLONELLA I. General Cultural, Ecological, and
613
Biochemical Considerations. J. Bacteriol. 1964. 87 (1), 162-170.
614
(33) Yang, Q; Xiong, P; Ding, P; Chu, L; Wang, J. Treatment of petrochemical wastewater by
615
microaerobic hydrolysis and anoxic/oxic processes and analysis of bacterial diversity.
616
Bioresour. Technol. 2015. 196, 169-175.
617 618
(34) Tsukamoto, T, K; Miller, G, C. Methanol as a carbon source for microbiological treatment of acid mine drainage. Water Res. 1999. 33 (6), 1365-1370.
619
(35) Fukui, M; Teske, A; Aßmus, B; Muyzer, G; Widdel, F. Physiology, phylogenetic
620
relationships, and ecology of filamentous sulfate-reducing bacteria (genus Desulfonema).
621
Arch. Microbiol. 1999. 172 (4), 193-203.
622
(36) Achenbach, L, A; Michaelidou, U; Bruce, R, A; Fryman, J; Coates, J, D. Dechloromonas
623
agitata gen. nov., sp. nov. and Dechlorosoma suillum gen. nov., sp. nov., two novel
ACS Paragon Plus Environment
Environmental Science & Technology
624
environmentally dominant (per) chlorate-reducing bacteria and their phylogenetic
625
position. Int J Syst Evol Microbiol. 2001. 51 (2), 527-533.
626
(37) Regueiro, L; Carballa, M; Álvarez, J, A; Lema, J.M. Enhanced methane production from
627
pig manure anaerobic digestion using fish and biodiesel wastes as co-substrates.
628
Bioresour. Technol. 2012. 123, 507-513.
629 630
(38) Gerardi, M, H. ORP management in wastewater as an indicator of process efficiency. YSI, Yellow Springs, OH. 2007.
ACS Paragon Plus Environment
Page 32 of 33
Page 33 of 33
Environmental Science & Technology
Graphical abstract Relative abundance of bacteria Biofilms on the inner pipe surface
100m
Firmicutes 17.40%
600m
Bacteroidetes 25.38%
Firmicutes 18.95%
Bacteroidetes 27.71% Proteobacteria 33.09%
Proteobacteria 44.11% Bacteroidetes 9.94%
1200m
Firm icutes 0.35%
Proteobacteria 72.01%
Variation of sewage quality along the sewer system
Proteobacteria Bacteroidetes Firmicutes Chlorobi Chloroflexi Spirochaetae Candidate_division_TM7 Others
Variation of biodegradable intermediates along the sewer system
ACS Paragon Plus Environment