Comparative Analysis on Microbial Characteristics of Aerobic
Activated Sludge and Biofilm in Low-temperature A
2
O Process
Wenai Liu
1,2 a
, Fan Wang
1,2 b
, Hua Kang
1,2 c
, Yiming Yan
1,2 d
and Dejun Bian
1,2,* e
1
School of Water Conservancy and Environmental Engineering, Changchun Institute of Technology, Kuanping Road,
Changchun, China
2
Key Laboratory of Urban Sewage Treatment of Jilin Province, Changchun, China
Keywords: The Low Temperature, Biofilm-Activated Sludge Coupling Process, High-Throughput Sequencing,
Microorganism.
Abstract: In the low-temperature A
2
O coupling system, activated sludge and biofilm perform corresponding
denitrification and phosphorus removal functions through the growth of specific microorganisms. The
difference of microbial populations under different growth conditions is of great significance to the
wastewater treatment effect. In this study, high-throughput sequencing technology was used to analyze the
population differences of suspended activated sludge and attached biofilms in aerobic cells in the same
environment, in order to analyze the pollutant removal mechanism. The results showed that the abundance
and diversity of aerobic biofilm fillers were higher than that of aerobic activated sludge, the microorganisms
in both biofilm and activated sludge mainly degrade organic matter. Both activated sludge and biofilm
accumulated and domesticated a variety of high-abundance denitrification and phosphorus removal bacteria,
but the distribution abundance of different bacteria in the two regions were different. The biofilm was rich
in many denitrifying bacteria Dokdonella, Denitratisoma, and phosphorus-accumulating bacteria
Candidatus_ Accumulibacter, and also enriched in Bacteroidetes_vadinHA17 with anaerobic phosphorus
removal function; While, the activated sludge contained more Cloacibacterium, Lactococcus related to
sugar metabolism, and enriched phosphorus accumulating bacteria Tetrasphaera,
Candidatus_Accumulibacter, and aerobic denitrifying bacteria Phreatobacter, Aeromonas.
1 INTRODUCTION
1
Biological treatment technology is widely used in
urban sewage treatment plants as a sewage
purification process with relatively low cost and
good treatment effect. The microorganisms in the
treatment system mainly achieve sewage purification
by metabolizing and decomposing organic pollutants
in the sewage (Zhang 2021). Therefore, the level of
microbial metabolic activity in the system directly
affects the sewage treatment effect. Among the
environmental impact factors, temperature is an
important factor (Dai 2020). In northern China, the
low temperature period in winter is long, which
a
https://orcid.org/0000-0002-2182-1604
b
https://orcid.org/0000-0001-8688-6102
c
https://orcid.org/0000-0002-1093-3168
d
https://orcid.org/0000-0003-3385-5497
e
https://orcid.org/0000-0001-6188-6560
seriously affects the degradation efficiency of
pollutants in the sewage treatment system (Shang
2020, Duan 2016). However, in practical
engineering and research, it has been found that the
biological treatment system still has a certain
removal effect on pollutants at low temperatures,
mainly because the cold-adapted microorganisms in
the system play a role (Shang 2020, Luo 2020).
Therefore, the microbial ecology analysis of the
changes in the microbial community structure and
the main functional flora and typical flora in sewage
treatment will help explain the pollutant removal
mechanism, thereby providing theoretical guidance
and technical support for the application of
biological improvement measures for low-
temperature operation.
In the low temperature environment in winter,
the performance of nitrification and phosphorus
removal is significantly reduced, and it is difficult
for a single treatment technology to make the
70
Liu, W., Wang, F., Kang, H., Yan, Y. and Bian, D.
Comparative Analysis on Microbial Characteristics of Aerobic Activated Sludge and Biofilm in Low-temperature A2O Process.
DOI: 10.5220/0011180600003443
In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 70-74
ISBN: 978-989-758-595-1
Copyright
c
2022 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
effluent of the sewage treatment plant reach the
sewage treatment standard. Microorganisms grow in
suspension in activated sludge and adhere to the
biofilm. Therefore, there is a big difference in the
groups of microorganisms that grow in two different
growth environments. The biofilm-activated sludge
coupling process adopts the combination of
activated sludge method and biomembrane method
to make up for the shortcomings of a single process,
which can effectively improve the removal effect of
pollutants and improve the stability of the system
(Ai 2021, Zhang 2021, Wang 2018). Studies have
found that the addition of fillers can effectively
improve the simultaneous removal of nitrogen and
phosphorus by the multi-stage AO process on
domestic sewage with a low carbon to nitrogen ratio
(Wang 2020).
In this study, high-throughput sequencing was
performed on the activated sludge and biofilm
samples in the aerobic zone of the biofilm-activated
sludge coupling A
2
O process, which worked well at
15℃. The differences in the community abundance
and structure of microorganisms growing in
suspension in activated sludge and adhering to
biofilms were analyzed to explain the removal
mechanism of pollutants in urban low-temperature
sewage treatment.
2 MATERIAL AND METHOD
2.1 The Reactor Devices
The experiment used a modified A
2
O reactor: the
aerobic zone was split into two parts of the same
volume; each part was filled with drifting balls, the
filling rate was 30%. The total reactor volume of
was 48 L, and the effective volume was 42 L,
divided into four tanks, and along the length of the
reactor were A
1
, A
2
, O
1
, and O
2
tanks. The volume
ratio of the anoxic tank to the aerobic tank of the
system reactor was 3:4; the interior of each tank was
filled with dispersing balls, and the inside of the
balls were equipped with polyurethane sponge and
Pall ring, the filling rate was 30%, and each
anaerobic tank was equipped with a stirrer Device.
2.2 Reactor Operating Conditions
The operating temperature was 15°C, simulating
low-temperature urban sewage. Adopting the
segmented water inlet method, the ratio of inlet water
flow rate was A
1
:O
1
=2:1, water inflow Q=84 L/d,
HRT=12 h, SRT=15 d, sludge reflux ratio 100%, and
the nitrification liquid reflux ratio was 200%.
2.3 Pollutant Removal Condition
The synthetic wastewater used in this experiment
simulated domestic sewage, including peptone, beef
extract, soluble starch, NH
4
Cl, KH
2
PO
4
,
CH
3
COONa, NaHCO
3
, etc. The experiment water
quality and effluent water quality when the reactor
was operated for 15 days were shown in table 1.
Table 1: Pollutant removal
water
quality
index
Concentration
range of influent
(mg/L)
Mean
concentration
of effluent
(
m
g
/L
)
Average
removal
rate
COD 246.80~328.70 33.36 90.9%
NH
4
+
-N 30.43~32.48 2.87 89.1%
TN 38.43~42.12 9.16 77.2%
TP 3.48~3.52 0.07 98.0%
2.4 High-Throughput Sequencing
Total genomic DNA of the biofilm and activated
sludge samples were extracted using the OMEGA
Soil DNA Kit (M5635-02) (Omega Bio-Tek,
Norcross, GA, USA) according to the manufacturer's
instructions.
The two-step PCR method was used to amplified
the extracted DNA, and the sample-specific 16-bp
barcode was incorporated into the forward and
reverse primers for multiple sequencing in the
second step of PCR. The total PCR amplicon was
purified by Agencourt AMPure Beads (Beckman
Coulter, Indianapolis, IN) and quantified using
PicoGreen dsDNA detection kit (Invitrogen,
Carlsbad, CA, USA). Finally, pair-end sequencing
was performed using the NovaSeq sequencer
platform from Shanghai Personal Biotechnology
Co., Ltd. (Shanghai, China). The analysis was
conducted on their online platform.
3 RESULTS AND DISCUSSIONS
3.1 Analysis of Microbial Abundance
and Diversity
The high-throughput sequencing results of activated
sludge and biofilm samples in aerobic cells showed
Comparative Analysis on Microbial Characteristics of Aerobic Activated Sludge and Biofilm in Low-temperature A2O Process
71
that the effective sequencing amounts of aerobic
sludge (Ae-S) and aerobic biofilm filler (Ae-B) were
75235 and 63873, respectively. The number of OTU
clusters of aerobic sludge and biofilm filler were
1258, 2085; 335 OTUs were owned by both, unique
OTUs for aerobic activivated sludge were 465, and
unique OTUs for biofilm were 697, represented by
Venn diagram as shown in Figure 1a. There were
much more microorganisms enriched in biofilm.
The OTU Rank diagram showed that the curve of
biofilm was wider and flatter, which indicating there were
more abundant microbial species and higher uniformity in
the biofilm (Fig. 1b). The results above indicated that
biofilm filler can enrich more kinds of microorganisms
and increase the biomass of the system.
(a) Venn diagram
(b) OTU rank diagram
Figure 1: Diagram of OTU distribution.
3.2 Analysis of Taxonomic
Composition
The microorganisms distribution in the aerobic
sludge and biofilm at different taxonomic level was
shown in Table 2. There were 22 phyla, 49 classes,
114 orders, 177 families and 263 genus
microorganisms detected in the aerobic sludge
sample; and 40 phyla, 85 classes, 177 orders, 277
families and 372 genus were detected in the biofilm
sample.
Table 2: Different levels of microbial taxa statistics
ID phylum class order family genus
Activated
Slud
g
e
22 49 114 177 263
Biofilm 40 85 177 277 372
The function of microorganisms largely depends
on the community structure of microorganisms. The
distribution the top 20 phyla with relative abundance
of samples in the sludge and biofilm of aerobic tank
was shown in Figure 2a. The results showed that
Proteobacteria had the highest abundance both in the
sludge and biofilm, followed by Bacteroidetes and
Chloroflexi in sludge, but there was more
Chloroflexi than Bacteroidetes in biofilm. The trend
of dominant bacteria at the phylum level for the two
samples was different.
The Figure 2b showed the top 20 bacterial genus
with relative abundance in the aerobic system. The
distribution of dominant bacteria at the genus level
in the sludge and biofilm of aerobic tank were also
very different as shown in Figure 2b. In activated
sludge, the bacteria that mainly related to sugar
metabolism (Han 2020) were Cloacibacterium
(6.38% of total sludge bacteria), Lactococcus
(3.18%); the bacteria that had phosphorus removal
function (Marques 2017) were Tetrasphaera
(6.33%), Candidatus_Accumulibacter (2.69%),
Acinetobacter (1.78%); the genus of bacteria with
denitrification function were Flavobacterium
(2.72%), Phreatobacter (2.53%), aerobic
denitrifying bacteria Aeromonas (1.78%),
Dokdonella (1.65%). The genus that playing roles in
aerobic biofilm were quite different from activated
sludge. The genus with sugar metabolism was
Candidatus_Competibacter (1.67% of total biofilm
bacteria); the genus that playing roles in phosphorus
removal were Candidatus_Accumulibacter (4.42%),
Bacteroidetes_vadinHA17 (2.02%) with the function
of anaerobic dephosphorization (Hao 2020); The
genus responsible for denitrification were
Dokdonella (4.80%) and Denitratisoma (2.55%).
The results showed that there were more long-
generation denitrification functional flora and some
other functional flora enriched in biofilm.
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
72
(a) phylum
(b) genus
Figure 2: Diagram of taxonomic composition.
3.3 Genus Composition Heat Map
In order to further compare the differences in species
composition between activated sludge and biofilm
samples, the data of the top 20 species with average
abundance was used to draw a genus composition
analysis heat map (Fig. 3). In the activated sludge
had gathered some bacteria related to flocculation,
such as Saccharimonadales, some aerobic
denitrifying bacteria, such as Aeromonas. And the
biofilm enriched with some anaerobic functional
microorganisms, such as Bacteroidetes_vadinHA17
had been reported to have the function of anaerobic
dephosphorization (Cho 2018), RBG-13-54-9 was
low-temperature dominant bacteria for phosphorus
removal
(Mei 2020). Under different carrier
environments, the distribution of genus-level
dominant bacteria in aerobic activated sludge and
biofilm was very different. This may be related to
the fact that the biofilm carrier could construct a
more complex environment.
Figure 3:
Diagram of genus composition heat map.
4 CONCLUSIONS
Although other conditions were the same, the results
showed that the abundance and diversity of
microorganisms on the biofilm were more than that
of activated sludge. The biofilm-activated sludge
coupling system increases the biomass in the
environment by creating more complex organisms
due to the addition of carriers. The biofilm was
enriched with more long-generation denitrification
functional flora, such as Dokdonella and
Denitratisoma, so adding biofilm to the activated
sludge system is more conducive to enhancing the
denitrification efficiency.
ACKNOWLEDGEMENTS
The research was funded by the Project of science
and technology development plan of Jilin Province
(20200201005JC), and the Project of science and
technology fund of Changchun Institute of
Technology (320210044).
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