Bioresource Technology 331 (2021) 125027

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

Bacterial community composition of internal circulation reactor at different

heights for large-scale brewery wastewater treatment
Junfeng Chen a, Yanyan Liu a, *, Kai Liu a, Lijun Hu a, Jiaqi Yang a, Xuemei Wang a,
Zhi-ling Song b, Yuewei Yang a, Meizhen Tang a, Renjun Wang a
a
Department of Environmental Science, School of Life Science, Qufu Normal University, Qufu 273165, PR China
b
Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, College of Chemistry and
Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR 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

• Bacterial community was analyzed in IC

reactor for brewery wastewater
treatment.
• Proteobacteria, Bacteroidetes, Chloroflexi
were dominant phyla in IC reactor.
• Longilinea, Desulfomicrobium, Caldithrix
and Geobacter were abundant genera.
• COD and BOD removal efficiency of IC
reactor were > 80% for brewery
wastewater.

A R T I C L E I N F O A B S T R A C T

Keywords: This study analyzed bacterial community structure for large-scale brewery wastewater treatment at different
Internal circulation (IC) reactor heights in internal circulation (IC) reactor. Proteobacteria, Bacteroidetes and Chloroflexi were dominant bacteria,
Bacterial community structure which accounted for 64.17%, 64.04%, 59.87% and 55.79% in phylum level, respectively. The unidentified
Brewery wastewater treatment
bacteria were accounted for a large proportion in genus level, available data showed that Longilinea, Desulfo­
Different heights
microbium, Caldithrix, Geobacter and Syntrophorhabdus were relatively abundant. Organic fermentation, hydro­
lysis, and acidification were mainly completed at the bottom, and production of hydrogen and methane were
completed in the upper and middle part of reactor. Alpha diversity and cluster distance analysis showed the
bacterial community could be divided into bottom, middle and upper part of IC reactor. The IC reactor possessed
the CODCr removal efficiency of 80% − 84.09%, and BOD5 of 77.50% − 86% for brewery wastewater. This study
would provide bacterial analysis references of IC reactor for industrial wastewater treatment in future.

1. Introduction which was in connection with the residual sugars remaining from pro­
cessing of the raw materials, as well as suspended solids, nitrogen
Brewery wastewater was characterized by high organic loading, element and phosphorus element, moreover, brewery wastewater

* Corresponding author..
E-mail address: liuyanyan@qfnu.edu.cn (Y. Liu).

https://doi.org/10.1016/j.biortech.2021.125027
Received 14 February 2021; Received in revised form 13 March 2021; Accepted 16 March 2021
Available online 27 March 2021
0960-8524/© 2021 Elsevier Ltd. All rights reserved.
J. Chen et al.
contained large amounts of glucose, maltose, maltotriose and dextrins
(Arantes et al., 2020). It was estimated that 3 ~ 10 L of brewery
wastewater will be produced for each liter of beer produced, if such large
amount of brewery wastewater was discharged directly, it would cause
serious water pollution and increase the environmental burden. The
major characteristics of brewery wastewater were of high chemical ox­
ygen demands (COD) for 20,000 ~ 60,000 mg/L, and the ratio of
biochemical oxygen demand (BOD)/COD were 0.5 ~ 0.7 (Jia et al.,
2020; Morita et al., 2011), which demonstrated that brewery waste­
water was full of good biodegradability and was suitable for biochemical
treatment.
The general treatment of brewery wastewater was anaerobic bio­
logical treatment, which converted the chemical energy stored in
organic compounds into simple substance and biogas (Jia et al., 2020;
Rao et al., 2007). The main processing treatment for brewery waste­
water were: anaerobic dynamic membrane bioreactor (MBR) (Alibardi
et al., 2016), upflow anaerobic sludge blanket (UASB) and digester
reactor (Chen et al., 2017a), anaerobic sequencing batch reactor (SBR)
combined with UASB reactor (Xu et al., 2019); aerobic biological
treatment, including aerobic activated granular sludge method (Corsino
et al., 2017); aerobic biological contactor method (Singh et al., 2019)
and the combination processes, such as UASB combined with traditional
activated sludge method (Cui et al., 2019; Song et al., 2020). Internal
circulation (IC) reactor was integrated by two UASB reactors vertically,
which has shown excellent performance of the 3rd generation anaerobic
bioreactors (Luo et al., 2016). Owing to its unique double-layer ­
structure and gas-driven internal circulation system, IC reactor was
characterized by larger height-diameter ratio, more effective bio­
mass retention and improved mixing intensity (Cui et al., 2011; Phan
et al., 2017). Based on these, IC reactor provided a promising choice for
the industrial wastewater treatment with high organic loading rate,
such as brewery wastewater. In normal conditions, organic loading rate
suited for IC reactors should be 3 ~ 5 times higher over UASB reactor
due to the better mass transfer because of the internal circulation of
intermixture (Jiang et al., 2014). A series of bacterial processes
including acidogenesis, acetogenesis and methanogenesis made up
anaerobic digestion, and these processes were controlled by acidogenic
bacteria, acetogenic bacteria and methanogenic archaea, respectively.
These bacteria were different widely from physiological characteristics,
nutritional requirements, growth dynamics and susceptible for envi­
ronmental changes (Perimenis et al., 2018). These bacteria played role
in interdependence and interaction in anaerobic digestion and kept
balance of these bacteria, which made anaerobic digestion process full of
vitality (Hao et al., 2012).
The good mass transfer between bacteria in sludge and substrates
worked in pollutants removal and methane production in anaerobic
reactor (Ni et al., 2020; Zhang et al., 2018). Enhanced the performance
of IC reactor with hydraulic circulation were always committed to the
researches in previous studies (Wang et al., 2014), however, compre­
hensive assessment on the effects of external circulation in the sludge
degradation kinetics and bacterial community analysis in real industrial
wastewater were lack of relevant studies. Moreover, the studies of rev­
ocatory external hydraulic circulation on the pollutants removal dy­
namic and bacterial community were relatively few in high organic-
loaded IC reactor. Bacteria played key role in pollutants removal and
good operation, and some studies have done lots of work in this area.
Qiu et al. (Qiu et al., 2013) studied the berberine reduction from
wastewater and the effects of berberine on bacterial community dy­
namics by a lab-scale UASB – MBR reactor, the removal efficiency of
berberine, COD and ammonia were reached up to 99%, 98% and 98%,
respectively, and Acinetobacter sp., Clostridium sp., Propionibacterium sp.,
and Sphingomonas sp. were dominant in UASB reactor. Chen et al. (Chen
et al., 2020a) studied the performance and bacterial community varia­
tions of a UASB reactor for treating monosodium glutamate wastewater
by increased organic loading rate, which demonstrated that the optimal
organic loading rate was 8 g (COD)/L/d, maximum COD removal
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Bioresource Technology 331 (2021) 125027
efficiency of 97%, and methane production rate of 2.3 L/L/d, and
Methanosarcina, Methanobacterium and Firmicutes were dominated in
UASB reactor after long term operation. However, the relationship be­
tween bacterial community analysis and reactor operation in real in­
dustrial wastewater treatment were relatively rare. The natural
properties of brewery wastewater can provide ideal conditions for
anaerobic digestion (Abdullah et al., 2016; Parawira et al., 2005).
However, promoting the growth of anaerobic bacteria and retaining
biomass remain significant challenges. The bacterial community struc­
ture analysis in IC reactor at different heights for large-scale brewery
wastewater treatment was full of significance.
In this study, high throughput sequencing was operated for under­
lying bacterial mechanisms at different heights under long-term opera­
tion of IC reactor. The basic operation taxonomy units (OTUs) analysis,
alpha diversity analysis, taxonomic analysis and beta diversity analysis
were conducted at different heights under of IC reactor. This study
would provide analysis for IC reactor from the perspective of bacterial
community structure in order to better understand the IC reactor, and
adjust the IC reactor from the perspective of bacterial community
structure to make it play a better effect.
2. Materials and methods
2.1. Overview of IC reactor
In this brewery, the brewery wastewater produced by the beer pro­
duction line was 4000 m3/d, the sewage treatment station was
composed of collection tank, regulating tank, acidification tank, IC
reactor, intermediate sedimentation tank, contact oxidation tank, in­
clined plate sedimentation tank. The designed influent CODCr and BOD5
concentration of the sewage treatment station was 6000 mg/L and 2000
mg/L, and the effluent CODCr and BOD5 concentration was 150 mg/L
and 60 mg/L. Faced with such high design requirements, the IC reactor
needed to play key role in the removal of COD and BOD concentration,
and the bacterial community structure in the IC reactor determined the
wastewater treatment capability of the IC reactor. Part of the water
discharged after being treated by the sewage treatment station was used
for plant greening of the brewery, and part of it was discharged to the
municipal sewage treatment plant. The IC reactor was a core part of the
brewery wastewater treatment station, and the schematic diagram of IC
reactor was shown in Fig. 1. The capacity of IC reactor to treat brewery
wastewater was 120 m3/h, the specifications (diameter and height) of
the IC reactor were ф11 × 21 m, the designed influent CODCr concen­
tration of IC reactor was 2000–2200 mg/L, and the effluent CODCr
concentration was 350–400 mg/L.
2.2. Sample collection and DNA extraction
Anaerobic sludge was sampled after 2 years of operation in the IC
reactor, and samples were taken at different heights of 2.2 m, 4.2 m, 6.2
m and 8.2 m by using reserved sampling tube pipes, respectively. The
obtained four samples were named as A1, A2, A3 and A4. DNA was
extracted by using a kit (Omega, USA), after the extraction of genomic
DNA was completed, the extracted genomic DNA was detected by 1%
agarose gel electrophoresis (Chen et al., 2020b, 2019a, 2019b).
2.3. PCR amplification and pyrosequencing
The 16S universal primers 357F and 518R were used for amplifying
the V3 region of the 16S rRNA gene, the polymerase chain reaction
(PCR) amplification mixture, and the 1st and 2nd PCR thermal cycling
programs has been described in detail in our previous studies (Chen
et al., 2017b). Three replicates were set for each sample, and after the
amplification was completed, the PCR products of the same sample were
mixed and detected by 2% agarose gel electrophoresis. The PCR prod­
ucts were recovered by AxyPreP DNA gel kit and then were eluted with
J. Chen et al.
Fig. 1. The schematic diagram of IC reactor.
Tris HCl and detected by 2% agarose gel electrophoresis again (Chen
et al., 2017d). After building the Miseq library, amplicons were
sequenced on the PE300 Miseq platform.
2.4. Bioinformatics, water quality and statistical analysis
Bioinformatics software of Trimmomatic, Usearch, Mothur and RDP
Classifier were used to filter the raw pyrosequencing data, cluster the
qualified sequences into OTUs with similarity cutoff by 97% analyze the
alpha diversity and the phylogenetic affiliation for each 16S rDNA
sequence. and principal coordinate analysis (PCoA) was conducted by R
software. Water quality analysis of CODCr and BOD5 were measured by
dichromate (K2Cr2O7) method and inoculation method (Chen et al.,
2017c, 2019b). Similarly, three replicates were conducted to ensure the
accuracy of the data for each test.
3. Results and discussion
3.1. Bacterial community structure analysis in IC reactor at different
heights
3.1.1. Overview of the pyrosequencing data
A total of 198,783 raw sequences were obtained, and 183,885 clean
sequences were achieved after quality control for the four samples. The
clean sequences for A1, A2, A3 and A4 were 49,389, 52,028, 44,614 and
37,854, respectively. The average length of the four samples were
distributed among 400–440 bp after quality control, which proportion
was 99.18% of the whole valid sequences. The richness rarefaction
curves and Venn analysis of the unique and shared OTUs of
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Bioresource Technology 331 (2021) 125027
pyrosequencing at genetic distance of 3% were shown in Fig. 2. The
trend of the four samples nearly reached saturation from the richness
rarefaction curves, which demonstrated the validity of sequencing re­
sults, and A2 showed more richness than other there samples prelimi­
narily with the random sampling method in terms of the obtained
sequences. The generated total OTUs were 1,432 for the four samples,
and the OTUs belonged to A1, A2, A3 and A4 were 1,061, 1,203, 1,151
and 1,095, respectively. There were 716 OTUs shared by the four sam­
ples, and 35, 29, 27 and 30 OTUs were exclusively found in A1, A2, A3
and A4. The shared OTUs between A1 and A2, A2 and A3, A3 and A4, A1
and A3, A2 and A4, A1 and A4 were 933, 1,008, 916, 881, 964, 859,
which demonstrated that the greater the height difference, the greater
the difference between the sequencing results preliminarily.
3.1.2. Bacterial community structure in phylum level
The classified 1,432 OTUs were clustered into 20 phyla for the four
samples, and the relative abundance in phylum level was shown in
Fig. 3. The main phyla in the four samples were Proteobacteria (22.85%
− 32.70%), Bacteroidetes (16.62% − 16.88%), Chloroflexi (12.55% −
24.57%), Firmicutes (6.07% − 8.94%).WS6, Synergistetes, Spirochaetae,
Thermotogae, Actinobacteria, Parcubacteria and Acidobacteria were also
found but at a relatively low ratios in the four samples. Proteobacteria,
Bacteroidetes and Chloroflexi were three dominant bacteria in phylum
level at different heights. The ratio of Proteobacteria in A2 (32.70%) was
more than that in other 3 groups (A1: 22.85%, A3: 30.69%, A4:
23.20%). Proteobacteria was the most abundant bacteria in lots of re­
actors in treating high concentration industrial wastewater. The results
demonstrated that Proteobacteria played key role in brewery wastewater
treatment, and the high relative abundance of Proteobacteria contributed
J. Chen et al.
Fig. 2. (a) The richness rarefaction curves of pyrosequencing at genetic distance of 3%; (b) The Venn analysis of the unique and shared OTUs of pyrosequencing at
genetic distance of 3%.
Fig. 3. The relative abundance for the four samples in phylum level in IC rector at different heights.
to the high degradation of organic matters in IC reactor (Guo et al.,
2018). The ratios of Bacteroidetes in the four biosamples were almost the
same, which demonstrated Bacteroidetes was stable in different heights
in IC reactor. Bacteroidetes took a key role in degrading refractory
organic matter, particularly in the removal of volatile fatty acids and
aquatic humic substances in IC reactor (Phan et al., 2017). The ratio of
Chloroflexi in A1 was 24.57%, which was more than that in other 3
groups (A2: 14.67%, A3: 12.55%, A4:15.71%). Chloroflexi was reported
to be related with degradation of organic matter in wastewater treat­
ment, which could be responded with the decreasing of COD concen­
tration (Ouyang et al., 2019; Yangin-Gomec et al., 2017). The ratio of
Firmicutes in A4 (8.94%) was largest, and A1 (6.07%) was the smallest of
the four groups, which demonstrated that the number of Firmicutes
increased with the increased of height along with this trend. Firmicutes
was related with the degradation of linear alkylbenzene sulfonate under
anaerobic conditions, while linear alkylbenzene sulfonate was regarded
as impeding methane production and persist in anaerobic treatment
technology (Li et al., 2015). The ratio changes demonstrated that linear
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Bioresource Technology 331 (2021) 125027
alkylbenzene sulfonate in brewery wastewater was mainly degraded by
Firmicutes, and this might be the reasons of methane produced in the
upper part of IC reactor. The trend of WS6, Synergistetes, Spirochaetae
and Thermotogae were similar as Firmicutes. WS6 were obligate fer­
menters and most likely be related with hydrocarbon fermentation in IC
reactor (Chang et al., 2019). Synergistetes could use amino acids from the
breakdown of proteins and peptides by other organisms to provide short-
chain fatty acids and sulfates such as methanogens and sulfate-reducing
bacteria for terminal degraders (Ouyang et al., 2019). Spirochaetae and
Thermotogae were related with the consumption and production of
hydrogen in anaerobic treatment in IC reactor (Yang et al., 2020). The
ratio changes of the 5 phyla seemed to show that the gas production
(methane and hydrogen) generated in upper part were more than that in
the bottom of IC reactor. The ratio of Actinobacteria in A3 and A2 (4.48%
and 2.70%) were more than that in A4 and A1 (2.28% and 1.58%), while
the role of Actinobacteria were the major drivers of hydrolysis acidifi­
cation in brewery wastewater (Liu et al., 2020), which reflected the
hydrolysis acidification in the middle part of IC reactor to some extent.
J. Chen et al.
The ratio changes of Parcubacteria were similar as Actinobacteria, its
main role was fermentation in brewery wastewater (Ma et al., 2020),
which also reflected the similar law. The ratio of Acidobacteria decreased
from the bottom to the upper part in IC reactor, and its role was
encoding a series of active enzymes to decompose, utilize and bio­
synthesize kinds of carbohydrates (Ma et al., 2020). Aminicenantes,
Caldiserica, Chlorobi and Fibrobacteres were also found in different height
in IC reactor but at lower level.
3.1.3. The alpha diversity analysis of bacterial community structure in IC
rector
The alpha diversity analysis of bacterial community structure was
characterized by Chao1 index, Observed species index, PD whole tree
index and Shannon index as shown in Fig. 4, and alpha diversity was
always used to evaluate the diversity in a certain area or ecosystem.
Chao1 index was used to estimate the bacteria richness (Fan et al.,
2018), the results showed that the bacteria richness of A3 and A2 were
higher than A4 and A1 and demonstrated that the middle part of IC
reactor possessed the greater diversity. Observed species index reflected
the actual number of OTUs included in the sample, the data showed that
A2 possessed the higher diversity than A3, A4 and A1, and reflected the
actual number of measured sequences in middle part was more than the
top and bottom part of IC reactor. PD whole tree index reflected the
difference in the preservation of evolutionary history among the species
in the sample, and the larger the index was, the greater the difference in
the preservation of evolutionary history among the species was, the
trend of PD whole tree index was similar as Observed species index, and
demonstrated similar rule of bacterial diversity in IC reactor. Shannon
index was used to assess the relative phylotype abundance in a certain
ecosystem, and the bigger Shannon index, the more abundant in
ecosystem (Chen et al., 2019b), the results showed that the abundant
order was A2 > A1 > A4 > A3, and indicated that the middle part of IC
reactor possessed the more abundant species and bacterial diversity. The
four indexes of alpha diversity analysis showed the same rule, that was
the middle part of IC rector possessed the more abundant and richness of
Fig. 4. The alpha diversity analysis of bacterial community structure in IC rector (a) Chao1 index; (b) Observed species index; (c) PD whole tree index; (d)
Shannon index.
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Bioresource Technology 331 (2021) 125027
bacteria, and more than the bottom and upper part of IC reactor.
3.1.4. Bacterial community structure in genus level in IC rector
The main relative abundance of the four samples in genus level in IC
rector at different heights was shown in Fig. 5. As shown in Fig. 5a, the
most major bacteria in genus level was unidentified, which were occu­
pied by 64.87%, 63.93%, 61.10% and 63.17%, which indirectly
demonstrated that it was a challenge to analyze the bacteria and it was
full potential to study bacteria in genus level deeply in IC reactor for
large-scale brewery wastewater treatment. For a clearer and more
intuitive analysis of the bacteria in genus level, the unidentified bacteria
were cut off in Fig. 5b. The ratios of Longilinea in A4 and A1 (6.09% and
4.15%) were larger than that in A2 and A3 (2.39% and 2.25%). Long­
ilinea was separated from methanogenic propionate-degrading consortia
and was a kind of strict anaerobe to ferment carbohydrates and generate
volatile fatty acid (Liu et al., 2017). The ratios of Desulfomicrobium in A1
and A2 (2.11% and 2.13%) were more than that in A3 and A4 (1.15%
and 0.74%), Desulfomicrobium could use hydrogen or simple organic
compounds (ethanol, pyruvate, and malate) as electron donors for sul­
fate respiration (Ouyang et al., 2019). The ratio of Caldithrix in A1
(2.00%) was far more than that in other 3 groups (0.66%, 0.44% and
0.52%). Caldithrix was a heterotrophic denitrifier and found in deep-sea
sediments in the anaerobic environment (Huang et al., 2019). The ratio
of Geobacter in A3 was 6.75%, which was more than that in A1 (1.72%),
A2 (2.10%) and A4 (0.69%). The role of Geobacter was the direct
interspecies electron transfer partners to donate electrons directly to the
relative methanogens (Wang et al., 2018). The ratios of Syntropho­
rhabdus were 1.60%, 1.42%, 0.90% and 1.23% in the four groups,
Syntrophorhabdus was related with methanogenic archaea (Vendruscolo
et al., 2020). The ratio of Weissella in A1, A2 and A3 were closely
(1.57%, 1.37% and 1.25%), which was more than that in A4 (0.42%).
Weissella was a kind of heterofermentative lactic acid bacterium, which
could produce ethanol, lactic and acetic acid (Si et al., 2015). The ratio
of Mesotoga in A4 was 2.56%, which was more than in A1 (1.36%), A2
(1.00%) and A3 (0.63%). The role of Mesotoga was making use of
J. Chen et al.
Fig. 5. The relative abundance for the four samples in genus level in IC rector at different heights (a: top 20 kinds of genus; b: top 19 kinds of genus and cutting out
the unidentified part).
organic small molecular substances as carbon and energy sources (Chen
et al., 2017a). The percentage of Christensenellaceae in A1 (1.17%) was
less than that in A2, A3 and A4 (2.72%, 2.22% and 2.20%). Christense­
nellaceae was regarded as strong metabolic activity for carbohydrates,
amino acids and carboxylic acids degradation, and its main function was
hydrolysis or acidogenesis (Wu et al., 2016). The ratio of Syntrophus was
fairly uniform among the 4 groups (1.11%, 1.04%, 0.93% and 1.02%).
Syntrophus was possessed of potential electroactivity in anaerobic
digestion and its function was interspecies for hydrogen or formate
transfer (Zhang et al., 2021). The ratio changes of these bacteria
demonstrated that the function of anaerobic treatment was reflected
throughout the whole IC reactor at different heights, however, there was
a trend that organic fermentation, hydrolysis, and acidification of
brewery wastewater were mainly completed at the bottom of IC reactor,
and the production of hydrogen and methane was completed in the
upper and middle part of the reactor. The percentages of Thauera,
Georgenia, Klebsiella and Microbacter in middle part of IC reactor were
relatively higher than those in the bottom and upper part, Thauera was
reported to be responsible for denitratation reaction process for brewery
wastewater treatment (Zhang et al., 2019); Georgenia mainly
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Bioresource Technology 331 (2021) 125027
contributed to the degradation of the carbohydrate fraction of brewery
wastewater (Chen et al., 2020c); Klebsiella was able to convert crude
glycerol to hydrogen at a high rate and yield (Chookaew et al., 2014);
the role of Microbacter was to produce volatile fatty acids in the treat­
ment of anaerobic processes for brewery wastewater (Cheng et al.,
2018). The ratio changes of these bacteria demonstrated that the func­
tion of anaerobic treatment was reflected throughout the whole IC
reactor at different heights, however, there was a trend that organic
fermentation, hydrolysis, and acidification of brewery wastewater were
mainly completed at the bottom of IC reactor, and the production of
hydrogen and methane was completed in the upper and middle part of
the reactor.
3.1.5. The cluster distance and PCoA analysis of bacterial community on
the similarity in IC reactor.
In order to explore the similarities of bacterial community structure
at different heights in IC reactor, the weighted heatmap of cluster dis­
tance and PCoA analysis were studied and shown in Supplementary
materials. Different colors represented different distances, the redder
the color, the closer the cluster distance, and the bluer the distance, the
J. Chen et al. Bioresource Technology 331 (2021) 125027

farther the cluster distance (Chen et al., 2019b). The results showed that Table 1
A2 and A3 shared much more similarities, and A2 and A3 were closer to The treatment effect of each processing unit.
A4 than A1, which demonstrated that the bacterial community structure Index Raw brewery Effluent of Effluent of Effluent of
shared more similarities in middle part, and shared more differences in wastewater acidification IC reactor aerobic contact
bottom and upper part with the height changes of IC reactor. PCoA was pool oxidation tank
performed by the vegan-package implemented in R software, the first CODCr 5000–6000 2000–2200 350–400 100–150
and second principal coordinate (PC1 and PC2) were occupied by (mg/
50.83% and 37.81%. The results showed that the bacterial community L)
BOD5 2000–2500 800–1000 140–180 60–80
structure was divided into 3 sections, A1 and A4 were independent, A2 (mg/
and A3 were in one aera, and demonstrated that A2 and A3 shared much L)
more similarities, which was far from A1 and A4, this rule was in line NH3-N 40–50 25–30 20–25 5–10
with the previous cluster distance. The weighted heatmap of cluster (mg/
L)
distance and PCoA analysis analyzed the bacterial community changes
TP (mg/ 10–15 10–15 10–15 1–2
at different heights in a view of Beta diversity, the bacterial community L)
shared much more differences in the bottom, middle part and upper part pH 6–10 6–10 6–10 6–10
with heights changes in IC reactor.

3.2. The performance of IC reactor for large-scale brewery wastewater 4. Conclusions

treatment
The entire brewery wastewater treatment plant was made up of
acidification pool, IC reactor and aerobic contact oxidation tank. The
treatment effect of each processing unit was shown in Table 1. It could
be seen that the removal of COD and BOD were mainly achieved by the
three units, and the removal of NH3-N and total phosphorous (TP) were
mainly achieved by aerobic contact oxidation tank. The influent and
effluent CODCr concentrations of IC reactor were 2000 – 2200 mg/L and
350 – 400 mg/L, and the CODCr removal efficiency was 80% − 84.09%.
The influent and effluent BOD5 concentrations of IC reactor were 800 –
1000 mg/L and 140 – 180 mg/L, and the BOD5 removal efficiency was
77.50% − 86%. The concentration changes of NH3-N and TP were not
obvious in IC reactor. The data demonstrated that the organic matters in
brewery wastewater was further fermented and utilized by different
bacteria in IC reactor, and the removal of nitrogen and phosphorus were
not obvious since there was no excess sludge discharge in IC reactor. The
role of IC reactor was combined acidification process and aerobic con­
tact oxidation process, it was like a bridge that combined the preceding
and the following for the degradation of organic matters, which was the
key and necessary process for brewery wastewater treatment.
3.3. Implications
IC reactor was not common in actual industrial wastewater treatment
in China. IC reactor was a connecting link between the preceding and the
following for the degradation of organic matters furtherly, and provided
a place for hydrogen and methane production. To meet and understand
IC reactor was the key to give full play to its role, while the bacteria
played a leading role for industrial wastewater treatment in IC reactor.
This study explored the bacterial community structure at different
heights for brewery wastewater treatment in IC reactor. Proteobacteria,
Bacteroidetes and Chloroflexi were dominant bacteria, followed by Fir­
micutes, WS6, Synergistetes, Spirochaetae, Thermotogae, Actinobacteria,
Parcubacteria and Acidobacteria in phylum level, and the role of each
bacteria were analyzed. However, the unidentified bacteria were
accounted for a large proportion, which indicated the lack of under­
standing and researching on IC reactor directly. The available data
indicated that Longilinea, Desulfomicrobium, Caldithrix, Geobacter, Syn­
trophorhabdus, Weissella, Mesotoga, Christensenellaceae, Syntrophus were
relatively abundant, followed by Thauera, Klebsiella, Aminivibrio, Geor­
genia and Microbacter in genus level at different heights in IC reactor.
The functions of these bacteria were also analyzed, they played an
important role in the further fermentation of organic matters and
methane production in treating brewery wastewater. Through our
limited research, the application range and the efficiency of IC reactor
were expected to expand, and made it better for the treatment of in­
dustrial wastewater to provide technical support.
This study analyzed bacterial community structure in IC reactor at
different heights for large-scale brewery wastewater treatment. Proteo­
bacteria, Bacteroidetes and Chloroflexi were dominant bacteria in phylum
level, which accounted for 64.17%, 64.04%, 59.87% and 55.79%,
respectively. In genus level, unidentified bacteria were accounted for a
large proportion, available data showed that Longilinea, Desulfomi­
crobium, Caldithrix, Geobacter, and Syntrophorhabdus were relatively
abundant, organic fermentation, and acidification were mainly
completed at the bottom, and methane production was completed in
upper and middle part. The IC reactor possessed CODCr removal effi­
ciency of 80% − 84.09%, and BOD5 of 77.50% − 86% for brewery
wastewater.
CRediT authorship contribution statement
Junfeng Chen: Conceptualization, Data curation, Formal analysis,
Funding acquisition, Investigation, Methodology, Project administra­
tion, Resources, Software, Supervision, Validation, Visualization,
Writing - original draft, Writing - review & editing. Yanyan Liu: Data
curation, Investigation, Methodology, Software, Validation, Writing -
original draft, Writing - review & editing. Kai Liu: Investigation, Vali­
dation, Writing - review & editing. Lijun Hu: Investigation, Validation,
Writing - review & editing. Jiaqi Yang: Investigation, Validation,
Writing - review & editing. Xuemei Wang: Investigation, Validation,
Writing - review & editing. Zhi-ling Song: Investigation, Validation,
Writing - review & editing. Yuewei Yang: Investigation, Validation,
Writing - review & editing. Meizhen Tang: Writing - review & editing.
Renjun Wang: Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
The authors were very grateful for the financial support provided by
Shandong Provincial Natural Science Foundation (ZR2020QC048 and
ZR2019BB040), the National Natural Science Foundation of China (NO.
31901188 and NO.31971503), Shandong Provincial Agricultural Fine
Species Project (2019LZGC020) and Experimental Teaching Reform
Research Project of Qufu Normal University (SJG201921).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.

7
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org/10.1016/j.biortech.2021.125027.
References
Abdullah, N., Fulazzaky, M.A., Yong, E.L., Yuzir, A., Sallis, P., 2016. Assessing the
treatment of acetaminophen-contaminated brewery wastewater by an anaerobic
packed-bed reactor. J Environ Manage 168, 273–279.
Alibardi, L., Bernava, N., Cossu, R., Spagni, A., 2016. Anaerobic dynamic membrane
bioreactor for wastewater treatment at ambient temperature. Chem. Eng. J. 284,
130–138.
Arantes, M.K., Sequinel, R., Alves, H.J., Machado, B., Fiorini, A., Silva, E.A.d., 2020.
Improvement of biohydrogen production from brewery wastewater: Evaluation of
inocula, support and reactor. Int. J. Hydrogen Energy 45, 5216–5226.
Chang, H.M., Sun, Y.C., Chien, I.C., Chang, W.S., Ray, S.S., Cao, D.T.N., Cong Duong, C.,
Chen, S.S., 2019. Innovative upflow anaerobic sludge osmotic membrane bioreactor
for wastewater treatment. Bioresour Technol 287, 121466.
Chen, C., Liang, J., Yoza, B.A., Li, Q.X., Zhan, Y., Wang, Q., 2017a. Evaluation of an up-
flow anaerobic sludge bed (UASB) reactor containing diatomite and maifanite for the
improved treatment of petroleum wastewater. Bioresour. Technol. 243, 620–627.
Chen, H., Wei, Y., Liang, P., Wang, C., Hu, Y., Xie, M., Wang, Y., Xiao, B., Du, C., Tian, H.,
2020a. Performance and microbial community variations of a upflow anaerobic
sludge blanket (UASB) reactor for treating monosodium glutamate wastewater:
Effects of organic loading rate. J. Environ. Manage. 253, 109691. https://doi.org/
10.1016/j.jenvman.2019.109691.
Chen, J., Hu, Y., Zhang, L., Huang, W., Sun, J., 2017b. Bacterial community shift and
improved performance induced by in situ preparing dual graphene modified
bioelectrode in microbial fuel cell. Bioresour Technol 238, 273–280.
Chen, J., Liu, S., Yan, J., Wen, J., Hu, Y., Zhang, W., 2017c. Intensive removal efficiency
and mechanisms of carbon and ammonium in municipal wastewater treatment plant
tail water by ozone oyster shells fix-bed bioreactor − membrane bioreactor
combined system. Ecol. Eng. 101, 75–83.
Chen, J., Liu, Y., Yang, Y., Tang, M., Wang, R., Jiang, L., Tian, Y., Hu, H., Zhang, X.,
Wei, Y., 2020b. Bacterial community structure and gene function prediction in
response to long-term running of dual graphene modified bioelectrode
bioelectrochemical systems. Bioresour Technol 309, 123398.
Chen, J., Yang, Y., Liu, Y., Tang, M., Wang, R., Tian, Y., Jia, C., 2019a. Bacterial
community shift and antibiotics resistant genes analysis in response to
biodegradation of oxytetracycline in dual graphene modified bioelectrode microbial
fuel cell. Bioresour Technol 276, 236–243.
Chen, J., Yang, Y., Liu, Y., Tang, M., Wang, R., Zhang, C., Jiang, J., Jia, C., 2019b.
Bacterial community shift in response to a deep municipal tail wastewater treatment
system. Bioresour. Technol. 281, 195–201.
Chen, J., Zhang, L., Hu, Y., Huang, W., Niu, Z., Sun, J., 2017d. Bacterial community shift
and incurred performance in response to in situ microbial self-assembly graphene
and polarity reversion in microbial fuel cell. Bioresour. Technol. 241, 220–227.
Chen, L.Y., Qin, Y.J., Chen, B.Q., Wu, C.L., Zheng, S.H., Chen, R.L., Yang, S.H., Yang, L.,
Liu, Z.J., 2020c. Enhancing degradation and biogas production during anaerobic
digestion of food waste using alkali pretreatment. Environ. Res. 188, 109743.
Cheng, D.L., Ngo, H.H., Guo, W.S., Chang, S.W., Nguyen, D.D., Kumar, S.M., Du, B.,
Wei, Q., Wei, D., 2018. Problematic effects of antibiotics on anaerobic treatment of
swine wastewater. Bioresour Technol 263, 642–653.
Chookaew, T., O-Thong, S., Prasertsan, P., 2014. Biohydrogen production from crude
glycerol by immobilized Klebsiella sp. TR17 in a UASB reactor and bacterial
quantification under non-sterile conditions. Int. J. Hydrogen Energy 39 (18),
9580–9587.
Corsino, S.F., di Biase, A., Devlin, T.R., Munz, G., Torregrossa, M., Oleszkiewicz, J.A.,
2017. Effect of extended famine conditions on aerobic granular sludge stability in
the treatment of brewery wastewater. Bioresour Technol 226, 150–157.
Cui, M.-H., Sangeetha, T., Gao, L., Wang, A.-J., 2019. Efficient azo dye wastewater
treatment in a hybrid anaerobic reactor with a built-in integrated bioelectrochemical
system and an aerobic biofilm reactor: Evaluation of the combined forms and reflux
ratio. Bioresour Technol 292, 122001. https://doi.org/10.1016/j.
biortech.2019.122001.
Cui, P., Zhou, X., Zhang, Y., 2011. The Feasibility Study of Cotton Pulp Wastewater
Treatment with IC Anaerobic Reactor. Procedia Environ. Sci. 11, 686–692.
Fan, T., Sun, Y., Peng, J., Wu, Q., Ma, Y., Zhou, X., 2018. Combination of amplified rDNA
restriction analysis and high-throughput sequencing revealed the negative effect of
colistin sulfate on the diversity of soil microorganisms. Microbiol Res 206, 9–15.
Guo, K., Shang, Y., Gao, B., Xu, X., Lu, S., Qi, Q., 2018. Study on the treatment of soybean
protein wastewater by a pilot-scale IC-A/O coupling reactor. Chem. Eng. J. 343,
189–197.
Hao, L.-P., Lü, F., Li, L., Shao, L.-M., He, P.-J., 2012. Shift of pathways during initiation of
thermophilic methanogenesis at different initial pH. Bioresour. Technol. 126,
418–424.
Huang, W., She, Z., Gao, M., Wang, Q., Jin, C., Zhao, Y., Guo, L., 2019. Effect of
anaerobic/aerobic duration on nitrogen removal and microbial community in a
simultaneous partial nitrification and denitrification system under low salinity. Sci
Total Environ 651 (Pt 1), 859–870.
Jia, R., Sun, D., Dang, Y., Meier, D., Holmes, D.E., Smith, J.A., 2020. Carbon cloth
enhances treatment of high-strength brewery wastewater in anaerobic dynamic
membrane bioreactors. Bioresour Technol 298, 122547.
Jiang, J., Wu, J., Zhang, J., Poncin, S., Li, H.Z., 2014. Multiscale hydrodynamic
investigation to intensify the biogas production in upflow anaerobic reactors.
Bioresour Technol 155, 1–7.
8
Bioresource Technology 331 (2021) 125027
Li, W.c., Wang, C., Tian, Z., Zhang, H., Gao, Y., Zhang, Y., Yang, M., Li, Y.-Y.,
Nishimura, O., 2015. Anaerobic treatment of p-acetamidobenzene sulfonyl chloride
(p-ASC)-containing wastewater in the presence or absence of ethanol in a UASB
reactor. Int. Biodeterior. Biodegrad. 98, 81–88.
Liu, H., Zhang, B., Yuan, H., Cheng, Y., Wang, S., He, Z., 2017. Microbial reduction of
vanadium (V) in groundwater: Interactions with coexisting common electron
acceptors and analysis of microbial community. Environ Pollut 231 (Pt 2),
1362–1369.
Liu, J., Wang, C., Wu, K., Huang, L., Tang, Z., Zhang, C., Wang, C., Zhao, X., Yin, F.,
Yang, B., Liu, J., Yang, H., Zhang, W., 2020. Novel start-up process for the efficient
degradation of high COD wastewater with up-flow anaerobic sludge blanket
technology and a modified internal circulation reactor. Bioresour Technol 308,
123300.
Luo, G., Li, J., Li, Y., Wang, Z., Li, W.-T., Li, A.-M., 2016. Performance, kinetics behaviors
and microbial community of internal circulation anaerobic reactor treating
wastewater with high organic loading rate: Role of external hydraulic circulation.
Bioresour. Technol. 222, 470–477.
Ma, J., Yang, M., Shi, C., He, C., Yuan, Q., Li, K., Gong, H., Wang, K., 2020. Insight into
the benefits of anammox bacteria living as aggregates. Bioresour Technol 318,
124103.
Morita, M., Malvankar, N.S., Franks, A.E., Summers, Z.M., Giloteaux, L., Rotaru, A.E.,
Rotaru, C., Lovley, D.R. 2011. Potential for Direct Interspecies Electron Transfer in
Methanogenic Wastewater Digester Aggregates. mBio, 2(4), e00159-11.
Ni, B.J., Zeng, S., Wei, W., Dai, X., Sun, J., 2020. Impact of roxithromycin on waste
activated sludge anaerobic digestion: Methane production, carbon transformation
and antibiotic resistance genes. Sci Total Environ 703, 134899.
Ouyang, E., Lu, Y., Ouyang, J., Wang, L., Wang, X., 2019. Performance and dynamic
characteristics of microbial communities in multi-stage anaerobic reactors treating
gibberellin wastewater. J Biosci Bioeng 127 (3), 318–325.
Parawira, W., Kudita, I., Nyandoroh, M.G., Zvauya, R., 2005. A study of industrial
anaerobic treatment of opaque beer brewery wastewater in a tropical climate using a
full-scale UASB reactor seeded with activated sludge. Process Biochem. 40 (2),
593–599.
Perimenis, A., Nicolay, T., Leclercq, M., Gerin, P.A., 2018. Comparison of the acidogenic
and methanogenic potential of agroindustrial residues. Waste Manag 72, 178–185.
Phan, T.N., Van Truong, T.T., Ha, N.B., Nguyen, P.D., Bui, X.T., Dang, B.T., Doan, V.T.,
Park, J., Guo, W., Ngo, H.H., 2017. High rate nitrogen removal by ANAMMOX
internal circulation reactor (IC) for old landfill leachate treatment. Bioresour
Technol 234, 281–288.
Qiu, G., Song, Y., Zeng, P., Duan, L., Xiao, S., 2013. Combination of upflow anaerobic
sludge blanket (UASB) and membrane bioreactor (MBR) for berberine reduction
from wastewater and the effects of berberine on bacterial community dynamics.
J Hazard Mater 246-247, 34–43.
Rao, A.G., Reddy, T.S.K., Prakash, S.S., Vanajakshi, J., Joseph, J., Sarma, P.N., 2007. pH
regulation of alkaline wastewater with carbon dioxide: a case study of treatment of
brewery wastewater in UASB reactor coupled with absorber. Bioresour Technol 98
(11), 2131–2136.
Si, B., Li, J., Li, B., Zhu, Z., Shen, R., Zhang, Y., Liu, Z., 2015. The role of hydraulic
retention time on controlling methanogenesis and homoacetogenesis in biohydrogen
production using upflow anaerobic sludge blanket (UASB) reactor and packed bed
reactor (PBR). Int. J. Hydrogen Energy 40, 11414–11421.
Singh, R., Bhunia, P., Dash, R.R., 2019. Optimization of organics removal and
understanding the impact of HRT on vermifiltration of brewery wastewater. Sci
Total Environ 651 (Pt 1), 1283–1293.
Song, C., Hu, X., Liu, Z., Li, S., Kitamura, Y., 2020. Combination of brewery wastewater
purification and CO2 fixation with potential value-added ingredients production via
different microalgae strains cultivation. J. Cleaner Prod. 268, 122332.
Vendruscolo, E.C.G., Mesa, D., Rissi, D.V., Meyer, B.H., de Oliveira Pedrosa, F., de
Souza, E.M., Cruz, L.M., 2020. Microbial communities network analysis of anaerobic
reactors fed with bovine and swine slurry. Sci. Total Environ. 742, 140314.
Wang, C., Liu, Y., Gao, X., Chen, H., Xu, X., Zhu, L., 2018. Role of biochar in the
granulation of anaerobic sludge and improvement of electron transfer
characteristics. Bioresour Technol 268, 28–35.
Wang, J., Xu, W., Yan, J., Yu, J., 2014. Study on the flow characteristics and the
wastewater treatment performance in modified internal circulation reactor.
Chemosphere 117, 631–637.
Wu, Y., Wang, C., Liu, X., Ma, H., Wu, J., Zuo, J., Wang, K., 2016. A new method of two-
phase anaerobic digestion for fruit and vegetable waste treatment. Bioresour Technol
211, 16–23.
Xu, X., Martin, G.J.O., Kentish, S.E., 2019. Enhanced CO2 bio-utilization with a
liquid–liquid membrane contactor in a bench-scale microalgae raceway pond. J. CO2
Util. 34, 207–214.
Yang, H., Si, B., Huang, S., Liu, Z., Zhang, Y., 2020. Effect of pH control on biohythane
production and microbial structure in an innovative multistage anaerobic hythane
reactor (MAHR). Int. J. Hydrogen Energy 45 (7), 4193–4204.
Yangin-Gomec, C., Pekyavas, G., Sapmaz, T., Aydin, S., Ince, B., Akyol, C., Ince, O., 2017.
Microbial monitoring of ammonia removal in a UASB reactor treating pre-digested
chicken manure with anaerobic granular inoculum. Bioresour Technol 241,
332–339.
J. Chen et al.
Zhang, Y., Guo, B., Zhang, L., Zhang, H., Liu, Y., 2021. Microbial Community Dynamics
in Granular Activated Carbon Enhanced UASB Treating Municipal Sewage under
Sulfate Reducing Conditions. Chem. Eng. J. 405, 126957.
Zhang, Z.-Z., Cheng, Y.-F., Zhu, B.-Q., Liu, Y.-Y., Zhang, Q., Jin, R.-C., 2019. Achieving
completely anaerobic ammonium removal over nitrite (CAARON) in one single
Bioresource Technology 331 (2021) 125027
UASB reactor: Synchronous and asynchronous feeding regimes of organic carbon
make a difference. Sci. Total Environ. 653, 342–350.
Zhang, Z., Gao, P., Cheng, J., Liu, G., Zhang, X., Feng, Y., 2018. Enhancing anaerobic
digestion and methane production of tetracycline wastewater in EGSB reactor with
GAC/NZVI mediator. Water Res. 136, 54–63.