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Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Study on dyeing wastewater treatment at high temperature by MBBR

and the thermotolerant mechanism based on its microbial analysis
Chao Li a,b , Zhen Zhang b , Yi Li a,b , Jiashun Cao a,b,
a
b

Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, Hohai University, Nanjing 210098, China
College of Environment, Hohai University, Nanjing 210098, China

a r t i c l e

i n f o

Article history:
Received 23 June 2015
Received in revised form 3 August 2015
Accepted 9 August 2015
Available online xxx
Keywords:
Dyeing wastewater
MBBR
Microbial community
EPS
Thermotolerant mechanism

a b s t r a c t
The dyeing wastewater treatment under increasing high temperature condition was studied in MBBRs.
Results showed that as the temperature increase, the COD removal exhibited two best performances
at 40 C and 50 C, and the maximum NH3 -N removal was obtained at 35 C and 40 C. The optimum
temperature for extracellular polymeric substances (EPS) yield is 45 C and the humic acid was the major
contributor. Microbial community was analyzed by the association of DGGE, real-time PCR and highthroughput sequencing technologies. The highest abundance of AOB (characterized by amoA genes) was
obtained at 35 C, and it conrmed that the biolm (attached on llers) can facilitate the AOB abundance
maintaining in the sludge. The precursor and successor thermophilic communities were enriched and
dominant at different temperature stages, which mainly conclude genera Caldilinea (from 35 C to 45 C)
and genera Rubellimicrobium and Pseudoxanthomonas (over 50 C), respectively. It meant the thermophilic
community displayed great evolution at the critical temperature 45 C|50 C. Additionally, the process
of thermotolerant mechanism establishment of the sludge in the MBBRs is proposed and the two-stage
enrichment of different thermophilic communities was considered as the key procedure.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
The textile industry is one of the most important industries
whose produced wastewater is characterized by high COD, high
color and refractory organics [1]. The biological technologies, rather
than some physical and chemical treatments [2], are considered as
the most widely applicable method for dyeing wastewater treatment, because it is simple and cost-effective [3]. Most of the
present biological treatment for dyeing wastewater operated under
mesothermal condition (below 40 C). However, the discharge of
textile wastewater usually suffers from relative high temperature.
For example, the water temperature of pulp and bleaching efuent
for knitted products is about 4045 C, and the desizing scouring
wastewater for woven fabric can reach 50 C or even higher.
The dyeing wastewater with high temperature requires cooling prior to biological proceeding, or would greatly impact on the

Abbreviation: MBBR, moving bed biolm reactor; COD, chemical oxygen

demand; EPS, extracellular polymeric substances; DGGE, denaturing gradient gel
electrophoresis; PCR, polymerase chain reaction; AOB, ammonia oxidizing bacteria.
Corresponding author at: College of Environment, Hohai University, Nanjing
210098, China. Fax: +86 2583786701.
E-mail address: 27394574@qq.com (J. Cao).

efuent [4,5]. The cooling process would increase the cost and the
cooling equipment corrosion cannot be ignored. So, there is a dearth
of knowledge on dyeing wastewater treatment under the higher
temperature.
Thermophilic bacteria had been widely found [6] and used for
wastewater treatment years ago, such as brewery wastewater [7],
slaughter wastewater [8] and vegetable waste [9]. Comparing to the
mesothermal, thermophilic treatment possesses own higher pollutant degradation rates, with about 310 times higher [4]. Moreover,
thermophilic treatment possesses also combine with other benets (e.g., lower excess sludge production [10], or higher pathogenic
bacteria removal [11]).
However, the wastewater at high temperature has to face
new challenges in biological treatment. High temperature leads to
poor sedimentary property of the sludge, which is usually compensated by biolm system, in order to reduce the sludge loss
[12]. Another challenge is the severe stress for the microorganism growth. Although the research on microbial community earned
much attention at the high temperature [13], since microorganisms
play the key role in biological treatment, the establishment process
of thermotolerance mechanisms (as the temperature increase), is
still lack of systematic study.
In this study, the moving bed biolm reactor (MBBR) was applied
to treat dyeing wastewater, with the additional llers to help com-

http://dx.doi.org/10.1016/j.procbio.2015.08.007
1359-5113/ 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: C. Li, et al., Study on dyeing wastewater treatment at high temperature by MBBR and the thermotolerant
mechanism based on its microbial analysis, Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.08.007

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pensate for the poor sedimentary property of the sludge. Under the
aerobic condition, as the temperature increasing gradually from
30 C to 55 C in the MBBR, we aim to: (1) investigate the pollutants
removal efciencies in the dyeing wastewater at different temperature stages; (2) compare the microbial communities at temperature
stages, and propose the process of thermotolerance mechanism
establishment in the sludge as the temperature increasing gradually.
2. Material and method
2.1. Reactor and operation
The moving bed biolm reactors (MBBR) were constituted
by polymethyl methacrylate tanks (active volume 2 L), with the
polypropylene suspended ller (K1-type) addition in order to
enhance the occulation ability of sludge. In order to ensure the
repeatability, two identical MBBR reactors operated in parallel
(The results were obtained by averaged value of the two). Each
MBBR operated continuously, better agreed with the practical
application. The hydraulic retention time (HRT) of the reactors
was xed at 24 h and the dissolved oxygen (DO) was maintained
at 2.0 0.4 mg/L during the whole experiment, which conformed
to (simulated to) the practical process of the WWTP. The initial
sludge retention time (SRT) was about 15 days and the initial mixed
liquor suspended solids (MLSS) was 12,500 mg/L (the MLSS would
be reduced when the temperature increased over 35 C, Figure
S2).
The temperature was gradually increased stage by stage,
include 30 C (the original inoculated sludge), 35 C, 40 C,
45 C, 50 C and 55 C. Each stage was kept for at least 15
days after both the two MBBRs performance were stable
(characterized by COD removal efciency according to the efuent), and then the water temperature was increased to next
stage by 1 C/day, avoiding the shock of abrupt temperature
change.
2.2. Characterization and analysis of sludge properties
The raw wastewater was derived from the efuent of a dyeing company whose products majored in cotton woven fabric
and a part of jean (which leaded to high azo dyestuffs and
indigo dye in the wastewater), and was diluted (2-fold diluent) to COD 650 80 mg/L and NH3 -N 18 2.2 mg/L (the raw
wastewater was characterized by Table S1). The diluted inuent with relative low NH3 -N concentration (also the reactors
were equipped with covers) meant to prevent much volatilization of ammonia gas at high temperature by aerating (i.e. air
lift effect). The performance of the reactors was monitored
every day, including COD and NH3 -N, according to the Standard
Methods [14]. The obtained data were averaged by the two reactors.
2.3. Characterization of extracellular polymeric substances (EPS)
of the sludge
The EPS of the sludge at each temperature gradient (for suspended sludge only) was investigated. The EPS extraction process
was performed according to the protocol described [15], including
soluble EPS, loosely bound EPS and tightly bound EPS. The protein and carbohydrates contents were determined referred to the
previous reports [16].
Excitation-emission matrix (EEM) uorescence spectroscopy
was further used to analyze the EPS by the uorescence spectrophotometer (Hitachi, F7000), which includes the following
uorescence techniques: (i) excitation (EX) and emission (EM) were

both xed at 200600 nm, (ii) synchronous scan at a constant offset wavelength 5 nm between excitation and emission. The contour
map was obtained by Origin 8.0.

2.4. Microbial community analysis

The sludge samples at the end of each temperature stage were
collected (300 mg) and the total genomic DNA was extracted from
each sample by using the FastDNA kit (Q-Biogene, MP Biomedicals,
UK) as described in the manufacturers instructions. The extracted
DNA samples were stored at 20 C, preparing for the following
PCR-DGGE, real time PCR and High-throughput sequencing analysis.
The PCR of 16S ribosomal DNA genes (V3 region, short for 16S
genes) was performed for the total eubacteria investigation with
the primers 338f (5 -CCTACGGGAGGCAGCAG-3 , with GC-clamp
before DGGE) and 518 r (5 -ATTACCGCGGCTGCTGG-3 ), under the
following conditions: 94 C/5 min denaturation step; 30 cycles
of 94 C/30 s, 58 C/30 s, 72 C/45 s; and a nal extension step at
72 C/10 min.
DGGE was carried out in a denaturing gradient gel electrophoresis system for the puried products of PCR. Polyacrylamide
gels (10% (w/v)) were 18 cm 18 cm, thickness of 0.75 mm. The
electrophoresis was conducted in 1 TAE buffer at 60 C. The
condition of electrophoresis was 100 V, 12.5 h with denaturing gradients of 3565%. Gels were photographed using Kodak
1D Image Analysis Software after stained by ethidium bromide
(EB).
Quantication of AOB (nitrifying bacteria) was characterized by amoA genes targeted real-time PCR, with the primers
amoA-1F: GGGGTTTCTACTGGTGGT, amoA-2R: CCCCTCKGSAAAGCCTTCTTC [17]. The real-time PCR condition was: 900 s at 95 C,
40 cycles of 15 s at 95 C, 30 s at 63 C, 30 s at 72 C (also
for data acquisition step). While approximative quantication
of the total eubacteria amount was performed by 16S genes,
in order to act as the reference with the condition: 15 s
at 95 C, 30 s at 60 C for 40 times of repetition. One last
step from 60 to 95 C with an increase of 0.2 /s was added
to obtain a specic denaturation curve to ensure the accuracy of the amplication for all the real-time PCR assays
above.
Plasmids (pEASY-T1Cloning Kit, Transtaq) containing cloned
16S genes or amoA genes which were cloned to DH5 were
used to draw standard curves (r2 = 0.99 with amplication
efciency 100.5% for 16S genes; r2 = 0.997 with amplication efciency 97.1% for amoA genes). All the abundance data
of these genes were based on averaged 3 identical samples.
High-throughput sequencing of the 16S genes (Miseq) was
conducted for further analysis of the sludge samples at each temperature stage after 16S rRNA gene PCR amplication and PCR
products purication (for suspended sludge only). To amplify and
sequence the V1V2 hypervariable region of the 16S rRNA gene,
forward primer (50-AGAGTTTGATYMTGGCTCAG-30) and reverse
primer (50-TGCTGCCTCCCGTAGGAGT-30) were selected and different 8-bases barcodes and a Guanine were linked to the 50
end of each primer. Then the puried products were sent for
sequencing using Illumina sequencing platform (Miseq, Illumina
Inc., USA). The acquired data was processed by Sickle and Mothur
program to remove the low quality of sequence and reduce noises
(according to Q-score >25). Finally, the ltered sequences were
assigned to a taxon by the RDP classier. Additionally, the heatmaps
and cluster analyses were obtained using the R program (R3.1.0).

Please cite this article in press as: C. Li, et al., Study on dyeing wastewater treatment at high temperature by MBBR and the thermotolerant
mechanism based on its microbial analysis, Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.08.007

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promote metabolism rate which enhances the COD removal [4], and
also can enrich thermophilic bacteria during long time of acclimatization, which further contributes to the COD removal under higher
temperature.
It is noticed that the two maximum removals 69.8% and 70.1%
were obtained at 40 C and 50 C, but poor performance at the
intermediate 45 C (Fig. 1). It supposed that the thermotolerant
microorganisms which were responsible for COD removal before
40 C was eliminated gradually when the temperature further
increase. But the new thermotolerant community achieved enrichment when the temperature increased to 50 C. It is an indication
that the microbial community (responsible for COD removal) has
gone through great changes from 40 C to 50 C (see the discussion on microbial community below). Correspondingly, the NH3 -N
removal efciencies (the average value of the whole period at each
temperature stage) were 33.3%, 39.1%, 38.5%, 28.0%, 17.5% and
11.5%, respectively.
The relative low level of NH3 -N removal in this study probably resulted from the complex nitrogenous pollutants in dyeing
wastewater [19]. As seen in Fig. 1, higher temperature greatly
affected the nitrifying bacteria activity, for the nitrifying ability is
almost prevented over 42 C according to previous study [20]. The
remained NH3 -N removal over 50 C was probably caused by the
air lifting effect [21], even thought it has been prevented furthest in
this study (lower NH3 -N concentration, cover equipped and relative
low aerating).
The prole of NH3 -N removal exhibited rst increment and then
declination, and the maximum was obtained at 35 C, with a sharp
decrease at 4045 C, which agreed with the theoretical optimal
temperature 35 C of AOB activity [20]. However, at 40 C, the AOB
maintained the nitrifying ability to a certain extent, which probably
resulted from the protective effect by some thermophilic bacteria
enrichment.

Fig. 1. Performance of COD and NH3 -N removal during the whole experiment as
the temperature rise.

3. Result and discussion

3.1. Performance of the MBBRs and the temperature effect
analysis
3.1.1. Performance of the MBBRs at different temperature stage
Performance of the MBBR reactors (averaged data of the two),
during the whole experiment was obtained in Fig. 1, including
COD and NH3 -N removal efciency which we mainly focused on.
(The average COD and NH3 -N concentration of the inuent were
650 mg/L and 18 mg/L, respectively.)
Fig. 1 shows the COD and NH3 -N removal efciencies in the
MBBRs at each temperature stage (from 35 C to 55 C, and the 30 C
is the background value in the parent reactor for inoculation). The
stable COD removal efciency and lasting for 15 days was dened
as the end of each temperature stage.
According to Fig. 1, with the stepped increased temperature
from 35 C to 55 C, the COD removal efciencies (the average value
of platform period) were 60.7%, 63.2%, 69.8%, 54.2%, 70.1% and 41.5%
for 30 C, 35 C, 40 C, 45 C, 50 C and 55 C, respectively. The COD
removal rst increased gradually, and then decreased at 40 C|45 C,
but ascended again at 45 C|50 C. Finally, it dropped to very low
level over 50 C (further data over 55 C was not shown).
Obviously, in this study, higher temperature (such as 40 C and
50 C) can achieve relative higher COD removal, differing from some
previous study which stated COD removal would decrease as temperature rising [18]. It is because higher temperature usually can

3.1.2. Temperature effect analysis

Temperature is an important biological factor and affects many
biological processes. To better evaluate the (high) temperature
effect on the reactor performance, the temperature coefcient (Q10 )
which is a reaction parameter reecting the biological metabolic
rate variation by temperature effect in the bio-reactors, was
applied. Q10 is characterized by the rate of change in a biological
system as a consequence of increasing the temperature by 10 C
[22]:

Q10 =

 R  (T 10T
1

R2

1)

(1)

where R is the rate (here refers to COD or NH3 -N removal rate) and
T is the temperature.
The relationship between biological degradation of pollutants
and temperature, characterized by Q10 , were calculated by Eq.
(1): 0.92, 0.82, 1.66, 0.60 and 2.85 for each temperature interval
(30 C|35 C, 35 C|40 C, 40 C|45 C, 45 C|50 C, 50 C|55 C), while
0.72, 1.03, 1.89, 2.56 and 2.31 for NH3 -N removal. In the general
view, the value of Q10 indicates whether a biological metabolic
reaction is sensitive to the temperature uctuation. Therefore, the
NH3 -N removal was more sensitive to temperature, rather than
COD removal, based on their Q10 comparison. However, it is also
an indication that the biological process of COD removal can better adapt the high temperature stress due to the thermotolerant
mechanism establishment of the sludge. Only over 50 C, the COD
removal was easily affected by temperature (Q10 = 2.85 from 50 C
to 55 C).

Please cite this article in press as: C. Li, et al., Study on dyeing wastewater treatment at high temperature by MBBR and the thermotolerant
mechanism based on its microbial analysis, Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.08.007

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Fig. 2. The EPS composition analysis at each temperature stage from 30 C to 55 C

(The O refers to the inoculated sludge at around 30 C).

3.2. EPS characterization and sludge properties analysis

The EPS of the sludge samples at the 6 temperature gradients were characterized for analyzing the properties of the sludge
(Fig. 2).
The total amount of EPS (the total of soluble EPS, loosely bound
EPS and tightly bound EPS) was focused, and it rst increased and
then decreased, the maximum value 1748 mg/L was obtained at
45 C. The component of EPS in the sludge mainly included extracellular carbohydrate, protein and humic acid. The amount of protein
and humic acid exhibited the similar variation (Fig. 2), among of
which, the humic acid was dominant and took the main responsibility of the total EPS amount change.
The detailed variation of EPS component can be obtained
from the EEM uorescence spectroscopy at Fig. S1, which further
included the different fraction of EPS (i.e. soluble EPS, loosely bound
EPS, and tightly bound EPS), demonstrating the EPS variation exhibited consistency with the Fig. 2.
The EPS synthesis is considered as a self-protection of microorganisms under stress condition [23], including high temperature
[24]. In this study, the high temperature (when below 45 C) would
stimulate the EPS production, especially for the increasing of the
protein and humic aicd. It probably also resulted from the enrichment of some EPS high yield bacteria [25] (discussed below).
However, when the temperature further increased (higher than
45 C), it had an inhibitory effect on the production rates of all
fractions of EPS, or even damaged the cells.
Previous studies demonstrated that the optimum (relative
higher) temperature was 3540 C for some specic microorganisms on producing highest amount of EPS [24,26]. Nevertheless, in
this study, 45 C was the optimum temperature for EPS production. It is an indication that the stimulated EPS production was an
integrated effect of the whole microbial community, which would
increase the thermostability (by more EPS production) for the biological sludge. In fact, there is even higher optimal temperature
(55 C) for the active sludge EPS production [27].
3.3. Microbial community analysis comparison at different
temperature
3.3.1. PCR-DGGE analysis of microbial community
At the end of each temperature stage (the stable COD removal
efciency was obtained and lasted for 15 days), the microbial community in the sludge was considered to reach the homeostasis
(or approach steady state) [28], and the sludge sampled to be
microbial analyzed. The microbial community was rst analyzed

Fig. 3. The DGGE prole of each sample at different temperature stages. (O

referred to the original suspended sludge at around 30 C, S and B referred to
suspended sludge and biolm attached on the llers, respectively).

Table 1
The closest species and their classication of the representative DGGE bands.
No.

The closest species*

Classication

B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
B13
B14

Caldilinea aerophila DSM 14,535

Oscillibacter valericigenes Sjm18-20
Caldilinea tarbellica D1-25-10-4T
Bacillus sp. 1NLA3E
Nitrosomonas eutropha C91
Zunongwangia profunda SM-A87
Leptothrix cholodnii SP-6
Acidothermus cellulolyticus 11B
Geobacillus thermoglucosidasius C56-YS93
Gramella forsetii KT0803
Pseudoxanthomonas taiwanensis CB-225
Paracoccus denitricans PD1222
Geobacillus thermodenitricans NG80-2
Rubellimicrobium thermophilum C-lvk-R2A-2T

Chloroexi
Firmicutes
Chloroexi
Firmicutes
Betaproteobacteria
Bacteroidetes
Betaproteobacteria
Actinobacteria
Firmicutes
Bacteroidetes
Firmicutes
Alphaproteobacteria
Firmicutes
Alphaproteobacteria

by PCR-DGGE, for both suspended sludge and biolm (on llers)

samples from 35 C to 55 C (together with the original inoculated
suspended sludge, 30 C)
According to Fig. 3, great difference was observed in the microbial community structures among these samples, especially for the
comparison among different temperature stages, which indicated
the temperature effect took more responsibility in microbial community variation, rather than the llers effect. Additionally, at same
temperature stage, the biolm presented more detectable bands
than the suspended sludge (Fig. 3), which meant the llers enabled
the stratied structure of the sludge (biolm formation) and more
diverse microbial community was enriched.
Some representative bands were cut and sequenced for analyzing the characteristic microorganisms (listed in Table 1).
B1 and B3 were identied as Caldilinea aerophila and Caldilinea
tarbellica, belonged to the thermophilic genus Caldilinea, which can
use various carbohydrates [29]. These two bands exhibited specic
intense at 40 C and 45 C, but suddenly fainted (even disappear)
over 50 C according to Fig. 3, which meant the special enrichment
of Caldilinea at 4045 C.
B7 (Leptothrix cholodnii) and B8 (Acidothermus cellulolyticus) got
similar enrichment at 4045 C. Thereinto, B7 is a heterotrophic

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bacterium, also majored in 40 C and 45 C, whose formed lamentous sheath can exhibit potential bioremediation [30].
These above-mentioned bacteria (B1, B3, B7 and B8) which were
particularly enriched at 40 C and 45 C, would take the responsibilities for the high COD removal in this temperature range (the
rst peak in Fig. 1). With the temperature increase, the microbial community would display evolution and the original dominant
thermophilic community was eliminated and replaced by the new
species with more competitive advantage at higher temperature
(over 50 C).
B9, B11, B13 and B14 which gradually enriched as the temperature increase, or only appeared at 50 C and 55 C (Fig. 3).
They all were identied as the thermophilic bacteria, whose
closest phylogenetic species were Geobacillus thermoglucosidasius,
Pseudoxanthomonas taiwanensis, Geobacillus thermodenitricans
and Rubellimicrobium thermophilum, respectively [31,32,33]. These
species exhibited better adaptability at higher temperature and
would contribute to the satisfactory COD removal at 50 C and 55 C
(Fig. 1).
Additionally, B12 was a denitrier (Paracoccus denitricans),
reducing nitrite to N2 O [32], and the anoxic growth agreed with
its special enrichment in the biolm lanes (Fig. 3). B14 (R. thermophilum) was reported previously to be isolated from the colored
biolms [33], consistent with our study whose inuent was also
dyeing wastewater.
The B5 (Nitrosomonas eutropha) was a kind of AOB which only
can be observed at 35 C and disappeared soon as the temperature increase. The optimum growth temperature 35 C agreed with
the previous study [34] and the NH3 -N removal situation (Fig. 1).
Additionally, the particular enrichment of B5 in the biolm (rather
than the suspended sludge, Fig. 3), indicated the llers can help to
maintain nitrifying bacteria at relative higher temperature (35 C,
but helpless with much higher temperature).
B6 was identied as Zunongwangia profunda which can secrete
large quantity of EPS. Its high yield of EPS was conrmed to display
high viscosity and great tolerance to high temperature [25], so that
can protect themselves and other bacteria nearby.

3.3.2. The amoA genes quantication by real-time PCR

The quantication of amoA genes which has been extensively
applied for the quantication of ammonia oxidizers, was further
investigated.
The density of amoA gene ranged from 0.87 103 to
1.45 106 /mg sludge (Fig. 4), and the highest density was obtained
at the 35 C level, especially for the biolm. However, since the
MLSS (sludge) in the reactors (approximatively presented the total
bacteria amount) decreased gradually as the temperature increased
(Fig. S2), the relative abundance of amoA genes (characterized by
the ratio of amoA/16S) would be more responsible for the nitrication ability of the sludge (Fig. 4). The abundance 0.06% in the
inoculated sludge (about 30 C) increased to 0.43% at the 35 C
level, and then decreased to 0.08% at 40 C level.
The 40 C owned even higher amoA abundance than the 30 C,
since the abundance of other bacteria (total eubacteria) decreased
faster than AOB. It meant the activity of AOB at high temperature,
i.e. the thermotolerance of nitrifying ability has been enhanced.
Therefore, as the temperature increased (accumulation process),
the AOB achieved further enrichment and could maintain its (relative higher) abundance and nitrifying ability for a period of time.
Further, it can be noticed that the biolm can help enrich more
amoA genes (i.e. AOB), compared to the suspended sludge. This
result also agreed with the previous DGGE analysis (the particular
enrichment of B5, Fig. 3). Two reasons were probably responsible:
(1) the llers facilitated the contact between microorganisms and
oxygen, in addition, (2) the llers crashed into each other and cut

the bubbles into (more) micro-bubbles which promoted the oxygen

utilization efciency.

3.3.3. High-throughput sequencing (Miseq) for microbial

community analysis
High-throughput sequencing technology was applied to provide further evaluation of the microbial community. The suspended
sludge at each temperature stage was further investigated, because
according to the previous DGGE analysis (Fig. 3), the temperature
effect plays more important role in microbial community variation,
rather than the llers effect. The top abundant genera (>0.4% for at
least one sample) in each sample were selected (a total of 30 genera
for all 6 samples) and shown as base-10 logarithm, in the heatmap
(Fig. 5).
The biodiversities, characterized by ShannonWeiner index,
were 4.92, 5.04, 4.64, 3.45, 3.06 and 2.94 for inoculated sludge
(30 C), 35 C, 40 C, 45 C, 50 C and 55 C, respectively. In the
general view, the biodiversities decreased with the temperature
increasing. It demonstrated that high temperature condition would
eliminate many kinds of microorganisms, leading to lower biodiversity (more simplex microbial communities) [13].
The cluster analysis demonstrated that the high similarity
existed between 50 C and 55 C, and the close relationship among
35 C, 40 C and 45 C (Fig. 5). It meant that as temperature increase,
the great change of microbial community structure occurred at
4550 C.
According to their abundance, many thermophilic genera,
such as Rubellimicrobium and Pseudoxanthomonas began to be
enriched at 50 C, while others like Caldilinea was enriched from
35 C to 45 C, but was eliminated gradually at higher temperature (50 C). This is also consistent with the previous DGGE
results, which indicated that two groups of thermophilic microbial communitylower and higher were enriched and dominant
at different temperature stages. In addition, the thermophilic genus
Schlegelella [35] with high abundance in 50 C and 55 C, was further
found in miseq, rather than DGGE, whereas, it is on the contrary for the genus Geobacillus (found in DGGE but not miseq),
which meant these molecular technologies can be complementary.
In this heatmap (Fig. 5), there was no AOB, but NOB (include
Nitrospira and Nitrobacter) found whose abundance was >0.4%. The
poor AOB abundance agreed with the real-time PCR results (Fig. 4).
It is an indication that the NOB showed better thermostability
than AOB. And the abundance of NOB 0.83% (sum of Nitrospira
and Nitrobacter) decreased to 0.22% from 35 C to 40 C, and further exhibited sharp declination to 0.02% from 40 C to 45 C. The
variation trend agreed with the pre-existing study which stated
the rapid declination of NOB activity was obtained at (from) 42 C
[34].
In summary, many kinds of thermophilic species or genera,
belong to various classications, were enriched with the gradually increased temperature. These thermophilic communities
gradually evolved and acclimatized themselves to the changing (increasing) selective pressure, and contributed to the COD
removal in the reactors with higher temperature. Further, the thermophilic community also played the role in NH3 -N removal due
to their assimilation (directly), or led to a syngenetic growth with
nitrifying bacteria (indirectly), which helped maintaining relative
high NH3 -N removal ability under the stress of high temperature.
In this study, higher performance was achieved for dyeing
wastewater treatment (characterized by COD and NH3 -N removal)
at higher temperature condition (comparing to mesothermal condition), which mainly resulted from various thermophilic bacteria
enrichment in the sludge.

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Fig. 4. The amoA genes density and relative abundance of amoA (to 16S gene) at each temperature stage by real-time PCR.

3.4. The critical temperature effect on microbial community and

discussion on process of thermotolerant mechanism establishment
in the biological sludge
During the temperature gradually increase process, some critical temperature values (temperature change) should be concerned,
which greatly affected the microorganisms.
According to cluster analysis (Fig. 5), the microbial community structure showed great change from 45 C to 50 C. In other
words, the temperature critical value is 45 C|50 C for the microbial community structure variation, which was mainly caused by
different thermophilic community enrichment. Two taxa of thermophilic bacteria were enriched at different temperature range,

which can be grouped by the precursor at 3545 C level (lower)

and the successor at 5055 C level (higher). The lower group
mainly includes Caldilinea (according to miseq), or B1, B3, B7 and B8
(according to DGGE), whereas the higher contained genera Rubellimicrobium and Pseudoxanthomonas (according to miseq), or B9,
B11, B13 and B14 (according to DGGE). It demonstrated that the
great change of dominant thermophilic community occurred at the
45 C|50 C.
The 35 C|45 C is the critical temperature of nitrifying bacteria, among of which the AOB is 35 C|40 C (based on the results
of DGGE and real-time PCR), and the NOB is 40 C|45 C (based on
the results of miseq), similar to previous study [34]. However, the
40 C|45 C is another critical value for AOB in this study, accord-

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Fig. 5. The heatmap analysis of each suspended sludge sample at different temperature stages (shown as base-10 logarithm of the abundance). O referred to the original
inoculated sludge at 30 C.

ing to real-time PCR (and the NH3 -N removal situation), which

meant the thermophilic sludge (i.e. thermophilic community structure) could maintain the nitrifying ability (to a certain extend) at
higher temperature.
The 40 C|45 C is the critical temperature of the variation of
EPS yield, since the EPS amount sharply increased to the maximum
from 40 C to 45 C, and then also sharply decreased over 45 C
(Fig. 2).
In summary, the biological thermotolerant mechanism in the
sludge mainly includes the thermophilic community enrichment
and EPS variation, according to our study. Therefore, as the temperature increase, the whole process of thermotolerant mechanism
establishment in the sludge in MBBRs, is proposed as follows:
Before 40 C as the temperature increase, the rst group of thermophilic community (the precursor, with relative lower optimal
temperature) gradually enriches, which is dominant in the sludge
and responsible for the main COD removal. Meanwhile, the abundance of nitrifying bacteria reduced, among of which AOB is rst
eliminated and then for NOB. The biolm attached on the llers can
protect nitrifying bacteria from being eliminated to a certain extent.
From 40 C to 45 C, the EPS amount increases sharply to become
another contributor to the thermostability of the sludge (before
40 C, EPS slowly increases with the temperature), however, it
would decrease after 45 C. As the temperature further increases (to
the 5055 C stage), the second group of thermophilic community
(the successor, with relative higher optimal temperature) which
can acclimatize themselves to higher temperature achieves more
enrichment and replaces the rst group (the precursor) gradually.
The two-stage enrichment of different thermophilic communi-

ties plays the key role in microbial community evolution and the
thermotolerant mechanism establishment of the sludge.
The thermotolerant mechanism, as the temperature raise, is a
complex and integrated biological process, which is still lack of specic theory. Although only the practical dyeing wastewater is used,
the proposed thermotolerant mechanism process establishment
and the critical value of temperature, in this study, can provide
some theoretical reference for the future research with different
temperature in a more wide range (especially for dyeing efuent
treatment).

4. Conclusion
(1) During the temperature rising process from 30 C to 55 C, two
peak values of higher COD removal were obtained at 40 C
and 50 C, and the maximum nitrifying ability was observed
at 35 C and 40 C, in the MBBRs under aerobic condition (DO
2.0 mg/L).
(2) Two groups of thermophilic communities (precursor and successor) were enriched at different temperature stages, which
mainly conclude genera Caldilinea (from 35 C to 45 C) and
genera Rubellimicrobium and Pseudoxanthomonas (over 50 C),
respectively, i.e. the 45 C|50 C is the critical temperature of
the microbial community structure variation, especial for the
thermophilic community.
(3) The process of biological thermotolerant mechanism establishment was discussed as temperature increasing, which is
probably based on the two-stage enrichment of thermophilic

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communities and the EPS variation (rst increasing and then

decreasing) in the sludge.
Acknowledgements
This work was supported by the Major Science and Technology Program for Water Pollution Control and Treatment of China
(No. 2012ZX07101-003), the Natural Science Foundation of Jangsu
Province of China (BK20140852), and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education
Institutions.
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.procbio.2015.08.
007
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Please cite this article in press as: C. Li, et al., Study on dyeing wastewater treatment at high temperature by MBBR and the thermotolerant
mechanism based on its microbial analysis, Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2015.08.007