Comparative Analysis on Microbial Characteristics of Aerobic

Activated Sludge and Biofilm in Low-temperature A

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

School of Water Conservancy and Environmental Engineering, Changchun Institute of Technology, Kuanping Road,

Changchun, China

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

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

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

https://orcid.org/0000-0002-2182-1604

https://orcid.org/0000-0001-8688-6102

https://orcid.org/0000-0002-1093-3168

https://orcid.org/0000-0003-3385-5497

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

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

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

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

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

, A

, O

, and O

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

=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

Cl, KH

COONa, NaHCO

, 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

(

)

Average

removal

rate

COD 246.80~328.70 33.36 90.9%

-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

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

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

(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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