Bioresource Technology 179 (2015) 436443

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Bacterial community compositions of coking wastewater treatment

plants in steel industry revealed by Illumina high-throughput
sequencing
Qiao Ma, Yuanyuan Qu , Wenli Shen, Zhaojing Zhang, Jingwei Wang, Ziyan Liu, Duanxing Li, Huijie Li,
Jiti Zhou
Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology,
Dalian 116024, China

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Nine activated sludges from coking

wastewater treatment plants were
sequenced.
 Thiobacillus, Comamonas, Thauera,
Azoarcus and Rhodoplanes were the
core genera.
 Percentages of AOB and NOB were
low.
 Operation mode, temperature and
ow rate affected community
formation.

a r t i c l e i n f o a b s t r a c t

Article history: In this study, Illumina high-throughput sequencing was used to reveal the community structures of nine
Received 16 October 2014 coking wastewater treatment plants (CWWTPs) in China for the rst time. The sludge systems exhibited a
Received in revised form 10 December 2014 similar community composition at each taxonomic level. Compared to previous studies, some of the core
Accepted 12 December 2014
genera in municipal wastewater treatment plants such as Zoogloea, Prosthecobacter and Gp6 were
Available online 19 December 2014
detected as minor species. Thiobacillus (20.83%), Comamonas (6.58%), Thauera (4.02%), Azoarcus (7.78%)
and Rhodoplanes (1.42%) were the dominant genera shared by at least six CWWTPs. The percentages of
Keywords:
autotrophic ammonia-oxidizing bacteria and nitrite-oxidizing bacteria were unexpectedly low, which
Coking wastewater treatment plants
Illumina high-throughput sequencing
were veried by both real-time PCR and uorescence in situ hybridization analyses. Hierarchical cluster-
Bacterial community composition ing and canonical correspondence analysis indicated that operation mode, ow rate and temperature
Core population might be the key factors in community formation. This study provides new insights into our understand-
Nitrication ing of microbial community compositions and structures of CWWTPs.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
During the past several decades, the Chinese steel industry has
rapidly developed and become the worlds largest steel producer
Corresponding author. Tel.: +86 411 8470 6251; fax: +86 411 8470 6252.
E-mail address: qyy@dlut.edu.cn (Y. Qu).
since 1996 with 13.944.3% of the worlds steel production from
1996 to 2010 (China Iron and Steel Statistics Annual Report,
19962011). In contrast to the huge economic benets, environ-
mental problems, particularly coking wastewater stemming from
steelmaking processes, are becoming signicant. Coking wastewa-
ter is among the most refractory wastewater and contains large
quantities of inorganic pollutants and organic pollutants, such as

http://dx.doi.org/10.1016/j.biortech.2014.12.041
0960-8524/ 2014 Elsevier Ltd. All rights reserved.
 Q. Ma et al. / Bioresource Technology 179 (2015) 436443 437

ammonium, sulfate, thiocyanate, phenol and polycyclic aromatic
hydrocarbons (Guo and Fu, 2010; Kim et al., 2008). Conventional
biological treatments do not meet the new emission standard for
pollutants of the coking chemical industry (GB 16171-2012,
CODcr < 80 mg/L, NH4-N < 10 mg/L). Therefore, several novel
biological processes have emerged, such as anaerobicanoxic
oxicoxic (A/A/O/O) and anaerobicoxicanoxicoxic (A/O/A/O)
(Kim et al., 2008), which can further improve wastewater treat-
ment efciency.
Within the various treatment technologies, the functional sta-
bility of coking wastewater treatment plants (CWWTPs) primarily
relies on the dominant microbial activities and interactions within
highly diverse communities. The community structure, diversity
and uctuation will ultimately affect the performances of
CWWTPs, including the nutrient removal, toxin degradation and
sludge stability (Wagner and Loy, 2002). Many studies assessed
the microbial composition and its relationship to treatment perfor-
mance (Thomsen et al., 2007; Maneeld et al., 2005). However,
previous studies mainly focused on bioreactors or particular
bacteria, such as ammonia-oxidizing bacteria (AOB) and nitrite-
oxidizing bacteria (NOB), lacking an overview of the microbial
community features of CWWTPs. Furthermore, the low resolution
of conventional biological techniques, such as 16S rRNA clone
libraries and denaturing gradient gel electrophoresis, have pre-
cluded, even skewed the comprehensive characterization of
communities.
Recently developed high-throughput sequencing technology is
a highly efcient tool for identifying the entire prole of microbial
communities. In the eld of wastewater treatment, the rst com-
prehensive research of different wastewater treatment plant
(WWTP) activated sludges was conducted using the 454 pyrose-
quencing technique. It was found that different plants shared some
dominant and core genera, such as Zoogloea and Prosthecobacter
(Zhang et al., 2012). Similar results were also obtained by Wang
et al. (2012) and Hu et al. (2012). However, these studies mainly
focused on municipal activated sludge. A recent study indicated
that different industrial WWTPs exhibited distinct community
compositions and no core population was found among these
WWTPs (Ibarbalz et al., 2013). Deterministic factors, particularly
wastewater characteristics, act as the key factor in the community
assembly process. Therefore, it is hypothesized that there is a high
coherence of community composition for a certain type of waste-
water such as coking wastewater. A thorough understanding of
CWWTP community compositions will aid in developing promis-
ing strategies and proper management techniques for coking
wastewater treatment processes.
In this study, the activated sludges of nine CWWTPs from Chi-
nese steel companies were collected and sequenced using Illumina
high-throughput platform. The objectives were: (1) to reveal the
community diversity and structure, (2) to examine whether core
populations exist, and (3) to explore the relationships between
microbial community and environmental variables of CWWTPs.
2. Methods
2.1. Coking wastewater treatment plants and sample collection
Activated sludge samples were collected from the secondary
sedimentation tank of nine Chinese steel industry CWWTPs in
May 2013. The location and detailed information for each plant
are shown in Fig. S1 and Table 1. These plants performed different
operational processes, such as anaerobicoxic (A/O), anaerobic
anoxicoxic (A/A/O), anaerobicoxicoxic (A/O/O), A/A/O/O and
A/O/A/O. The steel group corporations were all the largest local
steel companies with steel productions of 37 million tons per
year. Triplicate samples for each plant were collected and stored
at 80 C before use.
2.2. DNA extraction and PCR
Genomic DNA of the 27 sludge samples was extracted using the
method of Purkhold et al. (2000) with minor modications. Briey,
the sludge was centrifuged and resuspended in 630 lL DNA-
extraction buffer. Then, it was successively treated with 60 lL lyso-
zyme mixture (37 C, 60 min), 60 lL protease mixture (37 C,
30 min) and 80 lL 20% sodium dodecyl sulfate (37 C, 90 min).
The mixture was extracted using phenolchloroformisoamyl
alcohol (25:24:1) at 65 C for 20 min, and the supernatant was
extracted using chloroformisoamyl alcohol (24:1). The extraction
was then mixed with 0.6 volumes of isopropanol and stored at 4 C
overnight. Finally, the DNA was obtained by centrifuging, washing
(70% cold ethanol), drying and resuspending in nuclease free water.
The DNA concentration was determined using a NanoDrop
(NanoDrop Technologies Inc., Wilmington, DE, USA) and Pico Green
assays. Primers for sequencing were 515F (50 -GTG CCA GCM GCC
GCG GTA A-30 ) and 806R (50 -GGA CTA CHV GGG TWT CTA AT-30 ),
with different barcodes for the V4 region of 16S rRNA gene
(Bates et al., 2011). The PCR was performed at 94 C for 1 min;
35 cycles of 94 C for 20 s, 53 C for 25 s, and 68 C for 45 s; and
a nal extension at 68 C for 10 min using the AccuPrime High
Fidelity Taq Polymerase (Invitrogen, Grand Island, NY, USA). The

Table 1
Characteristics of the nine CWWTPs.

Treatment Location Name COD (mg/L) NH4-N (mg/L) Flow rate Treatment T pH DO MLSS
Plant (m3/h) process (C) (mg/L) (mg/L)
Inuent Efuent Inuent Efuent
CW1 Shanghai Baosteel Group Corp. 2500 250 300 15 375 A/O/A/O 23 NAa NA NA
CW2 Zhangjiagang, Jiangsu Shagang Group 6000 150 250 20 300 A/O 24.5 7 36 4000
Jiangsu Co., Ltd. 5000
CW3 Jinan, Jigang Group Co., Ltd. 3800 400 250 20 120 A/A/O/O 26 78 24 NA
Shandong
CW4 Xingtai, Hebei Xingtai Iron & Steel 6000 200 200 10 28 AO/O 24.5 7 5 2500
Corp., Ltd.
CW5 Tangshan, Tangshan Iron & Steel Co. 3800 210 200 15 180 A/A/O/O 26 7 4 NA
Hebei Ltd.
CW6 Taiyuan, Taiyuan Iron & Steel 4000 150 300 20 200 A/A/O 27 NA NA NA
Shanxi Group Co. Ltd.
CW7 Benxi, Liaoning Benxi Steel Group Corp. 2500 150 200 40 30 A/A/O 27 7 5 4500
CW8 Benxi, Liaoning Benxi Steel Group Corp. 4500 300 100 30 70 A/A/O 27 7 46 4500
CW9 Benxi, Liaoning Benxi Steel Group Corp. 3500 150 100 10 130 A/A/O 27 78 36 2500
a
NA: date are not determined.
438 Q. Ma et al. / Bioresource Technology 179 (2015) 436443
PCR products were pooled and puried using the QIAquick Gel
Extraction Kit (Qiagen, Valencia, CA, USA). Finally, the DNA library
was constructed and run on the Miseq Illumina at the Institute for
Environmental Genomics (IEG), University of Oklahoma.
2.3. High-throughput sequencing data analysis
Paired-end reads were joined by Flash, and the low quality
sequences were then removed. Chimera was detected by UCHIME,
and the resulting high quality sequences were processed to gener-
ate operational taxonomic units (OTUs) by CD-HIT at the 97%
sequence similarity threshold. The taxonomic assignment was
performed with the RDP classier with a condence cutoff of 0.5.
Hierarchical clustering analysis was performed using CLUSTER
and visualized using TREEVIEW, and other statistical analyses were
performed with the IEG pipeline (http://ieg.ou.edu). The average
data were calculated for each treatment plant before analyzing
the unique and shared OTUs/genera. The gures were generated
with SigmaPlot 11.0 and Excel.
2.4. FISH analysis
The activated sludge samples were sonicated and washed using
phosphate-buffered saline (PBS, pH 7.2). Then it was xed with 4%
paraformaldehyde for 3 h at 4 C and stored in a 1:1 mixture of PBS
and ethanol at 20 C. The hybridization and washing procedures
were the same as the protocol reported by Manz et al. (1994).
Probes NSO190 (50 -CGATCCCCTGCTTTTCTCC-30 ) was used for
AOB. NIT3 (50 -CCTGTGCTCCATGCTCCG-30 ) and Ntspa662 (50 -GGAA
TTCCGCGCTCCTCT-30 ) were used for NOB. CNIT3 (50 -CCTGTGC
TCCAGGCTCCG-30 ) and CNtspa662 (50 -GGAATTCCGCTCTCCTCT-30 )
were the competitor probes for NIT3 and Ntspa662, respectively.
Total cells were stained by DAPI (40 ,6-diamidino-2-phenylindole)
for 15 min after hybridization. Fluorescent samples were examined
by a laser scanning confocal microscope (FV1000, Olympus, Japan).
The software ImageJ (http://rsbweb.nih.gov/ij/) was used for FISH
quantication and the proportions of AOB and NOB were deter-
mined as the average areas to that of the total cells (at least 10
views of each sample).
2.5. Real-time PCR analysis
All quantitative amplications were conducted in triplicate
using the SYBR Green Real-Time PCR Kit (Novland, Shanghai, China)
and respective primers. The real-time PCR (qPCR) assays were per-
formed on Mx3000P qPCR System (Agilent, Germany). Primers used
in this study were the same with that of Gmez-Silvn et al. (2014),
Fig. 1. Hierarchical clustering (A) and detrended correspondence analysis (B) of the nine different activated sludge communities at OTU level (triplicate samples for each
plant).
of which 16S rRNA gene was for total bacteria and AOB and nxrB for
NOB. DCT was calculated as DCT = CT(ref)  CT(target), and the propor-
tions of AOB and NOB were calculated as ratiotarget=ref 2DC T
(Zhang et al., 2009). It should be noted that the average 16S rRNA
copies for bacterial cell is 4.2, while AOB usually harbors one
copy of rRNA operon and Nitrospira is considered to have two copies
of nxrB (Gmez-Silvn et al., 2014).
3. Results and discussion
3.1. Overview of sequencing and microbial diversity
Illumina high-throughput sequencing was used to investigate
the microbial community diversity and structure of CWWTPs. After
removing low quality sequences and chimeras, at least 15,930
effective sequences were obtained for each sample with an average
length of 253 bp. The sequence number of each sample was
normalized and 352805 OTUs were generated using the CD-HIT
clustering method with a threshold of 0.97. Detrended correspon-
dence analysis (DCA) and hierarchical clustering analysis indicated
that the triplicate samples of each plant were clustered, which
veried that the sequencing results were reproducible and reliable
(Fig. 1). A relatively high proportion of OTUs (21.6238.64%) were
shared among the treatment plants (Table S1). Further analysis
showed that only 70 of 3014 OTUs were shared by all plants
(Table S2), whereas these OTUs accounted for 64.15% of the total
sequences. This indicated a high coherence of community among
these CWWTPs.
For each community, the Shannon indexes (H) were in the range
of 3.004.40 (lower H values, lower a-diversity), corresponding to
the low OTU values ranging from 365 to 776, and Chao1 values
from 534 to 1223 (Table S2). The diversity and richness of the
CWWTPs were similar to other industrial wastewater plants and
bioreactors, but noticeably lower than that of the municipal waste-
water treatment systems (Ibarbalz et al., 2013; Zhu et al., 2013;
Zhang et al., 2012; Xia et al., 2010). Although over 15 thousand
reads were obtained for each sample, the rarefaction curves did
not reach the plateau (Fig. S2), suggesting there were several minor
species remained unidentied.
3.2. Bacterial community analysis at high taxonomic levels
The primers 515F and 806R were universal for a broad range of
bacteria and archaea, which could yield accurate phylogenetic
information (Bates et al., 2011). In the present study, nearly all
the sequences (over 99.99%) were assigned to bacteria and only
few sequences belonged to archaea in total. Of the total sequences,
 Q. Ma et al. / Bioresource Technology 179 (2015) 436443 439

Fig. 2. Percentages of the major phyla in each treatment plant (the sequence percentage is above 1% in at least one treatment plant).

Table 2
Percentages of the major classes in each plant (%).

Class CW1 CW2 CW3 CW4 CW5 CW6 CW7 CW8 CW9
b-Proteobacteria 26.14 29.46 33.58 67.97 43.79 59.69 49.12 50.73 36.39
a-Proteobacteria 26.23 19.33 25.81 15.52 10.39 26.45 10.30 13.65 21.61
c-Proteobacteria 12.44 18.66 18.23 1.82 16.63 3.65 8.16 11.51 5.50
Clostridia 1.13 0.34 0.32 3.58 0.69 0.05 2.64 5.73 4.46
Sphingobacteria 2.53 0.80 0.81 0.25 12.48 5.29 3.57 3.25 8.23
Acidobacteria_Gp4 5.29 14.36 14.35 0.32 0.04 0.08 0.01 2.17 0.17
d-Proteobacteria 4.60 5.25 0.28 0.20 4.05 0.15 0.89 1.72 1.13
Planctomycetacia 1.14 1.07 0.45 0.33 0.20 0.94 1.72 0.88 1.78
Actinobacteria 1.70 0.83 0.80 0.40 0.33 0.75 10.05 0.74 5.52
Bacteroidetes_incertae_sedis 1.12 2.17 0.02 0.33 0.00 0.00 2.26 0.44 0.22
Nitrospira 0.36 0.30 0.17 0.01 0.01 0.71 3.37 0.01 0.76
Deinococci 1.41 0.30 0.55 0.02 0.18 0.01 0.05 0.01 0.00
Ignavibacteria 1.18 0.71 0.72 0.01 5.45 0.00 0.00 0.08 0.02
Minor 2.44 1.01 1.16 0.51 0.66 0.55 2.21 1.91 3.11
Unclassied 12.29 5.40 2.76 8.74 5.10 1.68 5.64 7.19 11.09

3.58% were not classied at the phylum level, and the major phyla
for each plant were shown in Fig. 2. The most abundant phylum
was Proteobacteria with 76.14% of the sequences on average (min-
imum 65.18% in CW9 and maximum 90.38% in CW6). Previously, it
was shown that the phylum compositions in different industries
were different, and Proteobacteria was the prominent phylum in
pharmaceutical, petroleum renery, pet food industrial wastewa-
ter treatment plants (WWTPs) and sewage (Ibarbalz et al., 2013).
Moreover, Proteobacteria was the dominant phylum, with 2153%
and 3665% in various municipal wastewater treatment plants
and wastewater treatment bioreactors (Zhang et al., 2012; Wang
et al., 2012; Wagner and Loy, 2002). Compared to the previous
reports, Proteobacteria occupied higher percentages in the steel
industrial wastewater in this study, and even reached 90% in
CW6. The following major phyla were Bacteroidetes (mean 6.79%,
each plant 0.8713.63%), Acidobacteria (4.32%, 0.1814.74%), Firmi-
cutes (2.40%, 0.105.79%), Actinobacteria (2.35%, 0.3310.05%) and
Planctomycetes (1.84%, 0.277.80%), which were also widespread
in other systems (Maneeld et al., 2005).
At the class level, 93.35% of sequences were assigned, and there
was considerable consistency among the plants (Table 2). b-Proteo-
bacteria was the largest class with sequence percentages ranging
from 26.14% to 59.69%. The percentages of a-Proteobacteria were
10.3026.45% and c-Proteobacteria were 5.5018.66%. b-Proteobac-
teria were highly versatile in pollutant degradation capacities and
detected in various bio-treatment systems such as phenol-contain-
ing wastewater, domestic wastewater and coking wastewater
treatment systems (Figuerola and Erijman, 2007; Wang et al.,
2012). a-Proteobacteria and c-Proteobacteria were also reported as
the major phyla elsewhere, and a-Proteobacteria exceeded b-Prote-
obacteria in some plants (Xia et al., 2010). The percentages of d-Pro-
teobacteria ranged from 0.15% to 5.25%, which were lower than the
other proteobacterial groups. Except for the subdivisions of Proteo-
bacteria, other major classes contained Clostridia (2.10%), Sphingo-
bacteria (4.13%), Acidobacteria_GP4 (4.09%), Actinobacteria (2.35%),
Planctomycetacia (0.94%), Bacteroidetes incertae sedis (0.73%),
Nitrospira (0.63%), Deinococci (0.28%) and Ignavibacteria (0.91%).
At the family level, 71.92% of the total sequences were afliated
with 158 identied families, and the most abundant families were
Hydrogenophilaceae (20.84%), Rhodocyclaceae (12.78%) and Com-
amonadaceae (7.41%) (Fig. S3). Rhodocyclaceae and Comamonada-
ceae were the core families in many wastewater treatment plants
which were reported to be responsible for denitrifying and aro-
matic degrading processes (Loy et al., 2005). Hydrogenophilaceae
contains two genera, Hydrogenophilus and Thiobacillus, and only
Thiobacillus was detected in this study. Other dominant families
were Chitinophagaceae (3.15%), Xanthomonadaceae (2.83%), Rho-
dobacteraceae (2.52%), Hyphomicrobiaceae (2.18%), Rhizobiaceae
(1.58%), Kineosporiaceae (1.57%) and Sphingomonadaceae (1.54%).
3.3. Identication of the core genera
There were a large proportion of sequences (20.6750.25%) not
assigned to any genera. The major unidentied sequences were
440 Q. Ma et al. / Bioresource Technology 179 (2015) 436443

Table 3 few unique sequences (0.28% and 0.07%) existed in one and two
Percentages of the shared genera and their corresponding sequences. plants, respectively (Table 3). The major genera in each group were
Numbers of Numbers of Percentage in Percentage in summarized in Fig. 3. There were 32 genera in total, accounting for
treatment plants genera genera (%) sequences (%) 88.03% of the assigned sequences.
9 53 13.49 90.42 Thiobacillus was the primary genus and represented half the
8 80 20.36 93.84 sequences in CW6 and CW7 (Table S4). A total of 27 OTUs were
7 103 26.21 97.21 identied as Thiobacillus, of which OTU_104 contributed 98.64%
6 131 33.33 98.13
5 163 41.48 99.04
and was identied as Thiobacillus denitricans (99% similarity) after
4 193 49.11 99.42 re-classication via BLAST. T. denitricans is an autotrophic faculta-
3 240 61.07 99.72 tive anaerobic bacterium known for its ability to couple denitrica-
2 303 77.10 99.93 tion to inorganic sulfur-compound oxidation (Beller et al., 2006).
1 393 100 100
Therefore, it is widely used for denitrication processes of ground-
water and industrial wastewater treatment. In addition, the genus
Thiobacillus is responsible for thiocyanate biodegradation, which is
analyzed by BLAST using the 16S rRNA sequence database, and the ubiquitous in thiocyanate containing wastewater treatment
closest bacteria were listed for each OTU (Table S3). Consistent systems (Felfldi et al., 2010; Zhu et al., 2013). The high concentra-
with other studies, these novel bacteria (likely uncultured bacteria) tions of nitrate nitrogen together with thiocyanate in CWWTPs
might play important roles in wastewater treatment processes and create an ideal niche for Thiobacillus spp.
were required comprehensive evaluation. With the development of Comamonas, another core genus, was a major component in
metagenome sequencing technologies, it is now possible to recon- eight CWWTPs. It is a versatile aromatic degrader for phenolics,
struct the genome of the dominant or even minor species from polycyclic aromatic hydrocarbons and heterocyclic aromatics, such
activated sludge communities (Albertsen et al., 2013). as indole, quinolone and carbazole. In addition, it is also a promis-
Only 53 genera were shared by all CWWTPs, whereas these gen- ing denitrier, as well as a nitrier, under various aeration condi-
era represented 90.42% of the total identied sequences, and only a tions (Chen and Ni, 2011; Felfldi et al., 2010). The percentages

Fig. 3. Percentages of the major genera in each treatment plant (the values are calculated as log10(sequence numbers + 1), M means the numbers of plants with the genus
sequence percentage above 1%).
 Q. Ma et al. / Bioresource Technology 179 (2015) 436443
of Comamonas were very low (almost all < 1%) in the municipal
sewage and other types of industrial activated sludges (Ibarbalz
et al., 2013; Zhang et al., 2012; Wang et al., 2012). Therefore, its
occurrence might be related to high concentrations of phenol,
polycyclic aromatics and ammonia.
Azoarcus and Thauera often occur together and are functionally
important denitriers in activated sludge. They represented for
11.77% and 6.09% of the sequences, respectively, among
which CW4 harbored a signicantly high percentage of Azoarcus
(53.99%). As previously reported, Azoarcus and Thauera were the
most abundant potential denitriers in 17 nitrogen removal plants
as detected by FISH and various oligonucleotide probes (Thomsen
et al., 2007). With exception of denitrication ability, Azoarcus
and Thauera were also involved in aromatic biodegradation pro-
cesses. For example, Mao et al. (2010) isolated three Thauera
strains from a coking wastewater treatment bioreactor that could
degrade phenol, methylphenol and indole. In a recent study, the
genus Thauera was identied as the most dominant microorganism
in a granular sludge system, and it was proven to harbor perfect
nitrication capacity with 99.8% NH4-N (50 mg/L) removal rate
within 48 h (Zhao et al., 2013). The potential nitrication ability
of these heterotrophic bacteria provides another plausible explana-
tion for ammonia removal in CWWTPs which awaits further
investigation.
Acidobacteria, particularly subgroups Gp1Gp4 and Gp6, repre-
sents approximately 20% of all bacteria in the soil while rare pure
cultures are available (Naether et al., 2012). With the help of cul-
ture-independent technologies, Acidobacteria were frequently
detected in activated sludge, particularly Gp4 and Gp6, which were
the core genera shared by dozens of wastewater treatment systems
analyzed by pyrosequencing (Zhang et al., 2012; Wang et al.,
2012). In the present study, Gp4 was also observed as the major
constituent in CW1 (5.29%), CW2 (14.36%), CW3 (14.35%) and
CW8 (2.17%), whereas Gp6 rarely appeared in these plants (0.04%
on average). Limited knowledge is available for Gp6, thus novel
purication methods and metagenomic technologies are necessary
to provide information regarding this metabolically versatile
microbe (Jones et al., 2009). Other core genera included Kineospo-
ria, Ensifer, and Rhodoplanes, whereas the occurrence and role of
these bacteria were not frequently reported. There were some gen-
era only dominated in certain CWWTPs, such as Steroidobacter
(8.25%, CW5), Sphingobium (1.75%, CW3) and Rhizomicrobium
(1.94%, CW2). The community compositions of several municipal
wastewater treatment systems have been analyzed by 454 pyrose-
quencing, of which the genera Zoogloea, Dechloromonas, Ferruginib-
acter, Prosthecobacter, Gp6 and Subdivision 3 genera incertae sedis
were shared as the core populations (Zhang et al., 2012; Wang
et al., 2012). Notably, these species were almost undetectable in
the present study.
3.4. AOB and NOB analysis
Nitrication failure frequently occurs due to the high concen-
tration of ammonia and severe environmental conditions in coking
wastewater. Therefore, biological nitrication has attracted exten-
sive attention (Thomsen et al., 2007). As well known, nitrication is
generally carried out by two sequential steps, autotrophic ammo-
nia oxidation to nitrite by AOB, and autotrophic nitrite oxidation
to nitrate by NOB, which are the two important types of nitriers
(Siripong and Rittmann, 2007; Kim, 2013; Zhang et al., 2011). Since
nearly no archaea was identied by sequencing analysis, the
contribution of ammonia-oxidizing archaea could be ruled out.
Surprisingly, the percentages of AOB and NOB were below 1%,
except for Nitrosomonas in CW3 (1.9%) and Nitrospira in CW7
(3.4%) (Fig. 4). AOB are postulated as the main contributors to
ammonia oxidation, of which Nitrosomonas and Nitrosospira are
441
Fig. 4. Sequence numbers of Nitrosomonas, Nitrosospira and Nitrospira in each
treatment plant.
the most important genera. Nitrosomonas is resistant to the chang-
ing environment and has a relatively high growth rate compared to
Nitrosospira. Therefore, Nitrosomonas is often detected as the dom-
inant AOB in WWTPs (Siripong and Rittmann, 2007; Bai et al.,
2012). In this study, similar results were obtained from CW1 to
CW5. For CW6, CW7 and CW8, Nitrosospira exceeded Nitrosomonas,
though at a low percentage, suggesting that the AOB composition
for wastewater treatment systems should be evaluated case by
case. Nitrobacter and Nitrospira are the two typical NOB often pres-
ent with Nitrosomonas in activated sludge. Their occurrences and
proportions are inconsistent in different studies. For example,
Kim et al. (2011) and Zhu et al. (2013) observed that Nitrobacter
exceeded Nitrospira in full-scale and lab-scale coking wastewater
treatment processes, due to its higher resistance to toxic com-
pounds, whereas an adverse phenomenon was observed by Ye
et al. (2011). However, no sequences were obtained for Nitrobacter
in the present study, suggesting that Nitrospira is the major NOB in
CWWTPs, although it has low abundance (0.63%). To validate the
sequencing result, qPCR assay was conducted to calculate the per-
centages of AOB and NOB to total bacteria (Table S5). The relative
abundances of AOB and NOB were in the range of 0.0031.66% and
0.010.06%, respectively. The qPCR quantication results were in
agreement with sequencing ones.
High-throughput sequencing, as well as qPCR test, provide
enough depth to reveal microbial changes quantitatively, whereas
some biases associated with DNA extraction and PCR processes
have been identied (Albertsen et al., 2011; Zhang et al., 2012).
Therefore, another PCR-independent analytical technique FISH
was applied to quantify AOB and NOB abundances. It was shown
that the FISH quantitative detection was in accordance with that
of sequencing result generally (Table S5). The highest AOB and
NOB abundances were 2.5% (CW3) and 4.0% (CW7), respectively,
corresponding to 1.9% (CW3) and 3.4% (CW7) identied by high-
throughput sequencing analysis. The average proportions of AOB
and NOB were 1.0% and 1.6%, which further conrmed that the
autotrophic nitriers were present as minor genera in the steel
industrial sludge.
The unexpected low percentages of AOB and NOB seemed to be
conicted with successful nitrication. However, similar phenom-
enon was also observed in related studies. Ye et al. (2011) found
that only 0.05% and 1.01% of the microbes were AOB and NOB in
a full-scale wastewater treatment plant in Hong Kong. Figuerola
and Erijman (2007) could hardly detected any AOB using a 16S
442 Q. Ma et al. / Bioresource Technology 179 (2015) 436443
Fig. 5. Canonical correspondence analysis (CCA) of bacterial community structures from 27 samples with respect to the major genera relative abundances (A) and six
measurable variables (B). Arrows indicate the direction and magnitude of measurable variables associated with bacterial community structures. Each circle represents the
bacterial community structure for each sample.
rRNA gene library and FISH in an oil renery industrial WWTP with
high nitrication efciency. Furthermore, in an aerobic granular
sludge system with high ammonium concentration, AOB were
absent in the DGGE dominant bands (Zhao et al., 2013). Nitrica-
tion is considered to be the synergistic effects of heterotrophic
and autotrophic bacteria, AOB and NOB. In CWWTPs, high concen-
trations of COD and organic carbon sources provide excess growth
substrates for the potential heterotrophic nitriers such as
Comamonas and Thauera aforementioned. They may outcompete
autotrophic bacteria and contribute more to ammonium removal.
However, the specic roles and nitrication mechanism of hetero-
trophic bacteria required further study. A thorough understanding
and well control of these functional nitrifying species were of great
signicance for process management.
3.5. The relationships between bacterial community and operation
processes
Canonical correspondence analysis (CCA) and hierarchical clus-
tering analysis were conducted to discern the possible correlations
between bacterial community structure and environmental vari-
ables. The CWWTP communities were divergent at different levels
as shown in Fig. 1A and Fig. S4. The cluster results at the class and
phylum levels were disorganized because of the high consistency
of communities at high taxonomic levels. The grouping patterns
at the genus and family levels were similar to the OTU level which
were divided into three groups. Group I contained one member,
CW4, which was the only one operated with the A/O/O mode. It
also had the lowest diversity, with a Shannon index of 2.56 and a
Simpson index of 0.29. CW1 operated with A/O/A/O, and CW2 with
A/O, clustered together forming Group II. The remaining plants
generated the largest branch of Group III, which were operated
with A/A/O and A/A/O/O modes. It was indicated that the operation
modes of the CWWTPs played important roles in community
formation, and the A/O/O was distinctly different from others.
From Fig. 5A, it could be inferred that the genera were associ-
ated with different CWWTPs. Azoarcus was highly related with
CW4 and Thiobacillus was conned to CW7. Six environmental
variables, i.e. inuent COD, efuent COD, inuent NH4-N, efuent
NH4-N, temperature, and ow rate were all signicant according
to the permutation test analysis (p < 0.05). The length of the arrows
in the CCA plot corresponds with the strength of the correlation
between variables and community structure. It was shown that
ow rate and temperature were the most two signicant variables
(Fig. 5B). It was reported that the sludge bacterial community com-
position and structure exhibited temporal variations, of which
temperature was the most deterministic factor affecting microbial
community assembly (Wells et al., 2011; Flowers et al., 2013). In
the present study, only a single sampling date point for each plant
was investigated. It could be speculated that the bacterial commu-
nity in CWWTPs sludge also shifted seasonally, and further
investigations are required to reveal the temporal dynamics of
the sludge community.
4. Conclusions
There was considerable consistency of the bacterial community
composition in different CWWTPs as determined by Illumina
high-throughput sequencing. Thiobacillus, Comamonas, Azoarcus,
Thauera, and Gp4 were the main genera widespread in most sludge
samples whereas autotrophic nitriers, such as AOB and NOB
unexpectedly existed as the minor species. The operation mode,
ow rate and temperature of CWWTPs should be the key factors
in shaping the community, and the microbial community from
A/O/O mode was distinctly different from others. The profound
understanding of CWWTP microbial communities in this study will
be helpful in developing efcient strategies for coking wastewater
treatment.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (No. 21176040), the Program for New Century
Excellent Talents in University (No. NCET-13-0077), and the
Fundamental Research Funds for the Central Universities (No.
DUT14YQ107). We thank Jizhong Zhou and Yujia Qin for Illumina
high-throughput sequencing technical help.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2014.
12.041.
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