Comparative Analysis of Microbial Community Struc-
ture of Anaerobic Granular Sludge and Aerobic Granular
Sludge Based on High-throughput Sequencing Tech-
nology
Zhen MIN1,2,3, Hai GU1,3*, Tiancheng HUANG1,3
1School of Life and Geographical Sciences, Kashi University, Kashi, Xin-
jiang Province, China
2Key Laboratory of Biological Resources and Ecology of Pamirs Plateau
in Xinjiang Uygur Autonomous Region, Kashi, Xinjian Province, China
3School of Mechanical Engineering, Nantong Institute of Technology,
Nantong, Jiangsu Province, China
Abstract: Granular sludge has a variety of microbial communities related to its biological com-
munity, which is widely used in the field of sewage treatment. In this paper, A high-throughput All Rights Reserved.
sequencing analysis method was used to study the microbial community structure in aerobic and
anaerobic sludge. The 16S rDNA sequence amplified by PCR was used for molecular biology
analysis. At the phylum level, Proteobacteria and Chloroflexi are relatively abundant in aerobic
granular sludge, while Chloroflexi, Euryarchaeota, and Bacteroidetes are dominant bacteria in
anaerobic granular sludge. At the genus level, there is a certain biological similarity between the
two types of sludge, which may be related to the growth conditions of the microorganisms in the
sludge, the sewage treatment process used, and the quality of the sewage water. Our results indi-
cate that there are many unknown microorganisms in granular sludge. It may be a potential
source of new microbial resources. At the same time, the study of microbial community structure
in granular sludge can provide some reference value for sewage treatment.
Keywords: Sewage treatment; Granular sludge; Microbial community structure; High-throughput
sequencing
DOI: 10.47297/taposatWSP2633-456902.20200106
Corresponding author: Hai GU, School of Mechanical Engineering, Nantong Institute of Technology, 14
Yongxing Road, Gangzha District, Nantong, Jiangsu Province, 226001,China, Email address:
****************.
Comparative Analysis of Microbial Community Structure of Anaerobic Granular Sludge and Aerobic Granular Sludge Based on High-throughput Sequencing Technology
Introduction: Granular sludge refers to a kind of microbial aggregate with high density, large
volume formed by the mutual aggregation of microorganisms in the biological treatment system
of sewage wastewater under certain environmental conditions. According to the different condi-
tions required for the growth of microorganisms, granular sludge is divided into aerobic and an-
aerobic types. The granular sludge formed under anaerobic and anoxic conditions is called an-
aerobic granular sludge, such as the sludge in the Upflow Anaerobic Sludge Bed (UASB). The
granular sludge formed under the condition of sufficient dissolved oxygen (DO) is called aerobic
granular sludge, such as the sludge in the sequential batch reactor (SBR). At present, aerobic
granular sludge and anaerobic granular sludge have been widely used in the treatment of domestic
sewage and industrial wastewater(Urban et al. 2007 ; Bilardi et al. 2020).The anaerobic granular
sludge sewage treatment system has limitations such as long start-up period, low efficiency in
treating low-concentration organic wastewater. Aerobic granular sludge has problems such as
large amount of surplus sludge and high operating cost. Therefore, it is necessary to study the mi-
crobial community structure and its main functional community in aerobic and anaerobic granular
sludge. To seek solutions to the deficiencies of the two granular sludge wastewater treatment sys-
tems.
1. Materials & Methods
All Rights Reserved.
(1) Sample collection and pre-treatment
Aerobic and Anaerobic granular sludge samples were collected from a petrochemical wastewater treatment plant in Jiangsu Province, China. It can The collected sludge sample was
centrifuged at 1000r / min for 3min at high speed, the upper layer liquid was discarded, placed in
a 2ml sterilization centrifuge tube, frozen at -4 ℃, and sent to Shanghai Sangon Biotech for
high-throughput sequencing.
(2) Methods
Adopt high-throughput sequencing analysis method. DNA from granular sludge samples is extracted and amplified using polymerase chain reaction (PCR) followed by microbial classifica-
tion and sequencing.
2. Results
(1) Bacterial Community Diversity
By analyzing the microbial community structure of aerobic and anaerobic granular sludge samples using MiSeq, the effective sequences of bacterial sequencing were 78520 and 70765, and
OTU were 5096 and 4077, respectively（Table 1）. The sequencing coverage index is 0.96, so the
sequencing results can truly reflect the characteristics of microbial diversity in the sample.
According to Shannon and Simpson index, Chao1 and ACE index, it can be seen that aerobic granular sludge samples have higher community diversity than anaerobic granular sludge samples,
while biomass is lower (Table 1).
Theory and Practice of Science and Technology
The total OTU number in the two sludge samples is 484, accounting for 5.6% of the OTU numbe. This indicates that these bacterial groups are shared by aerobic granular sludge and an-
aerobic granular sludge, indicating that different sludge treatment systems have certain biological
similarities. The unique OTUs in aerobic granular sludge and anaerobic granular sludge were
4612 and 3593 respectively, and the unique OTUs accounted for 53% and 41% of the total OTUs.
This indicates that nearly half of the bacteria in the two sludge samples are different. It is related
to the growth conditions required by the bacteria in the sludge.
(2) Bacterial Community Structure
reactor techA total of 43 bacterial phyla reads were detected in the two sludge samples. Among aerobic
granular sludge, Proteobacteria is the most abundant phylum, accounting for 27.59%.Followed by
Chloroflexi, accounting for 22.16%, Plantomytomytes accounted for 8.05%.In addition, Ignavi-
bacteriae (6.73%), Acidobacteria (6.39%) and Bacteroidetes (4.40%) were detected, of which 8.56%
were unclassified bacteria. Among anaerobic granular sludge samples, Chloroflexi is the most All Rights Reserved.
abundant fungus, accounting for 23.88%, followed by Euryarchaeota, accounting for 15.39%, and
Bacteroidetes accounting for 12.57%.In addition, Proteobacteria (7.64%), Firmicutes (3.59%) and
Aminicenantes (3.41%) were detected, and 20.73% of the bacteria were not classified.
At the genera level, the bacterial sequences in aerobic and anaerobic granular sludge are di-vided into 424 and 257 different genera. The proportion of unclassified bacteria detected in the
two sludge samples was 50.85% and 47.33%, respectively. This is due to the rich diversity of en-
vironmental microorganisms themselves, and there are a large number of biological groups that
may be unclassified. A considerable part of the bacteria in the aerobic granular sludge is not
matched to any group, its proportion is the largest (50.85%), followed by Ignavibacterium ac-
counted for 6.69%, followed by Aridibacter (3.65%), Bellellia (3.18%), Armatimonadetes_gp5
1.93%), Thermogutta (1.48%).The most abundant genus of anaerobic granular sludge is Meth-
anothrix, reaching 13.41%, followed by Levilinea (9,59%), and the proportion of unclassified mi-
croorganisms is 47.33%.Methanothrix, Levilinea, Ignavibacterium, Aridinacter, Belllinena,
Aminicenantes, genera incertae sedies, Leptolinea, Parcubacteria incertae sedis, Mesotoga, Ther-
mogutta, Armatimonadetes gp5, Litorilinea, Candidatus Clode were detected in two different
sludges. The results show that these fungi are common in sludge used for sewage treatment, and
different granular sludges have certain biological similarity, which may be related to the growth
conditions required by the sludge, the sewage treatment process adopted, and the sewage water
quality. At the same time, unsorted bacteria are the predominant bacteria in the two types of
sludge, which indicates that the aerobic and anaerobic granular sludge are rich in bacterial diver-
sity.
Comparative Analysis of Microbial Community Structure of Anaerobic Granular Sludge and Aerobic Granular Sludge Based on High-throughput Sequencing Technology
3. Discussion
This study demonstrated that the predominant bacteria in aerobic granular sludge are Prote-obacteria and Chloroflexi. Proteobacteria has the ability to degrade pollutants, such as COD and
NH3-N. Chloroflexi can promote sludge flocculation and degradation of macromolecular organic
matter, and has good biological phosphorus removal ability. The predominant bacteria in anaero-
bic granular sludge are Chloroflexi, Euryarchaecta and Bacteroidetes. Euryarchaecta has a strong
adsorption capacity and the ability to oxidize organic matter. Bacteroidetes has a strong metabolic
effect on complex organic matter, protein and lipid in sewage. From the characteristics of granular
sludge, it can be seen that when treating medium and low concentration organic sewage, aerobic
and anaerobic granular sludge treatment systems have good sewage treatment effect. But when
dealing with high-concentration organic wastewater, the anaerobic granular sludge sewage treat-
ment system shows obvious advantages.
Proteobacteria is the most important phylum in the aerobic granular sludge, which is con-sistent with the results of other studies found that the aerobic granular sludge community structure
distribution(Gao et al. 2016; Wang et al. 2012). Some studies have pointed out that Bacteroidetes,
Chloroflexi, Actinobacteria are also the main bacterial phylum found in aerobic granular sludge
species(Gao et al. 2016; Wang et al. 2012 ). Chloroflexi are mostly filamentous bacteria, and exist
in the form of floc skeleton inside the sludge fungus flocs, which promotes the sludge floccula-All Rights Reserved.
tion(Larsen et al. 2008), and has the ability to degrade macromolecular organic matter and good
biological phosphorus removal. The unclassified bacteria of aerobicand anaerobic granular sludge
accounted for 50.85% and 47.33% respectively, indicating that the microbial diversity in aerobic
granular sludge is more abundant and there are more incalculable microbial resources. In future
research, it is necessary to combine pure separation culture methods and other molecular biologi-
cal methods such as fluorescence in situ hybridization (FISH) and fluorescence quantitative PCR
(q-PCR) to verify and improve the 16S rDNA clone library flora in this study Structural research.
4. Conclusions
Compared with anaerobic granular sludge, aerobic granular sludge has more microbial diver-sity, but its biomass is less than anaerobic granular sludge. Nearly half of the microorganisms in
the two sludges are different. Analysis of the microbial community structure of different granular
sludges can help to more comprehensively understand the similarities and differences between the
predominant flora in two granular sludge and the bacterial flora between different sludges. From
an academic point of view, studying the microorganisms in sludge samples is also important for
understanding microbial ecology, and at the same time provides a theoretical basis for the effi-
cient treatment of wastewater.
Theory and Practice of Science and Technology
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