Study on Microbial Population Difference for the Treatment of

Domestic Sewage between Micro-Pressure Swirl Reactor (MPSR) and

SBR

Hua Kang

1,2 a

, Fan Wang

1,2 b

, Wenai Liu

1,2 c

, Xichao Wang

2,3 d

, Lubo Shao

1,2 e

and Dejun Bian

1,2,* f

School of Water Conservancy and Environmental Engineering, Changchun Institute of Technology, 395 Kuanping Road,

Changchun, China

Key Laboratory of Urban Sewage Treatment of Jilin Province, Changchun, China

Changchun Municipal Engineering & Research Institute Co., Ltd., 855 Kunshan Road, Changchun, China

*Corresponding Author

Keywords: Micro-Pressure Swirl Reactor (MPSR), Microbial Community Structure, High-Throughput Sequencing.

Abstract: The comparative study on the microbial community structure of activated sludge from micro-pressure swirl

reactor (MPSR) and SBR under stable operation was conducted by the 16S rRNA third-generation high-

throughput sequencing, which was sampled from simulated domestic sewage. The experimental results

indicated that the two reactors had great differences in the microbial community structure of activated sludge

due to the different circulating flow patterns under the same water quality and intermittent operation mode.

Compared with SBR, MPSR had more dominant bacteria phyla and some functional bacteria of higher relative

abundance such as Flavobacterium and Thiotrix, and simultaneously strictly anaerobic, strictly aerobic and

facultative anaerobic microbial species existed so as to produce higher species diversity and population

richness, which is accord with the polyphase theory of the reactor.

1 INTRODUCTION

In the urban sewage treatment system, activated

sludge is very important for the removal of pollutants,

and the microbial community structure will directly

affect the stability and treatment efficiency of sewage

biological treatment. Therefore, the diversity of

community structure and dominant bacteria are one

of the main indicators to evaluate the structure and

function of sewage treatment system (Tian 2020).

Many scholars pointed out that the activated sludge

of sewage treatment plant had high species diversity

and community richness, and the influent quality,

process composition and operating conditions have a

certain impact on the microbial community structure

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

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

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

https://orcid.org/0000-0001-5198-8037

https://orcid.org/0000-0001-9908-0335

https://orcid.org/0000-0001-6188-6560

of activated sludge (Ma 2021, Chang 2021, Li 2021).

However, there are few comparative studies on the

microbial community structure of activated sludge

with different process types.

MPSR is a new sewage treatment device with

anaerobic, anoxic and aerobic environments

coexisted, which has good organic matter removal,

nitrogen and phosphorus removal effects (Bian 2020,

Bian 2020). In this study, high-throughput

sequencing technology was used to study the

microbial community structure of MPSR and SBR in

order to provide a theoretical basis for the

optimization of Activated Sludge Method wastewater

treatment process performance and provide reference

for subsequent research.

Kang, H., Wang, F., Liu, W., Wang, X., Shao, L. and Bian, D.

Study on Microbial Population Difference for the Treatment of Domestic Sewage between Micro-pressure Swirl Reactor (MPSR) and SBR.

DOI: 10.5220/0011177100003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 31-35

ISBN: 978-989-758-595-1

2 MATERIAL AND METHOD

2.1 Test Devices

The test device was shown in Figure 1. The effective

volumes of reactors were both 36L. The size of the

SBR was 300mm long, 300mm wide and 500mm

high. MPSR included two parts, the main reaction

zone and micro-pressure zone. The main reaction

zone was a diameter of 900mm and 90mm wide, and

the micro pressure zone was 130mm long, 90mm

wide and 400mm high. The flow rate of MPSR

gradually decreased from outside to inside, and the

concentration of DO also showed an obvious gradient

change from outside (2.02mg/L) to inside (lower than

0.05mg/L) (Bian 2020). SBR sludge was sampled

about 200mm below the liquid level, and MPSR

sludge was sampled from the mixed liquid at the nine

points in Figure 1. The inoculated sludge was taken

from an aeration tank of a sewage treatment plant at

Changchun, and the initial sludge concentration of the

mixed liquid was 2000mg/L. After aeration culture,

the sludge was evenly divided into two reactors

(abbreviated as R).

(a)SBR (b)MPSR

Figure 1: Test devices

2.2 Experiment Water Quality

The synthetic wastewater was used to simulate

domestic sewage, which including nutrients and trace

elements required by microorganisms from beef

extract, peptone, starch and etc. The concentration of

main water quality indexes was shown in Table 1.

Table 1: Influent water quality.

Index Range Average

COD/mg·L

-1

305.2~386.0 338.1

-N/mg·L

-1

29.8~36.4 31.4

TN/mg·L

-1

32.1~38.6 34.2

TP/mg·L

-1

2.5~4.5 3.2

2.3 Operating Condition

The two reactors were operated for two cycles every

day, with cycle time of 12h including 8h of aeration

(initial 5min of feeding), 3h of sedimentation, 10min

of drainage and 50min of free time. The aeration

capacity was 1.5L/min, and the operating temperature

was (20±1) ℃. The sludge residence time (SRT) was

22d, and the drainage ratio was 0.5. The two systems

operated stably for 30 days. During the operation, the

average removal rates of COD, NH

-N, TN and TP

of SBR were 92.0%, 98.8%, 66.3% and 95.8%

respectively, while 94.0%, 98.8%, 75.6% and 98.6%

in MPSR.

2.4 High-throughput Sequencing

Total genomic DNA samples were extracted using the

OMEGA Soil DNA Kit (M5635-02) (Omega Bio-

Tek, Norcross, GA, USA), following the

manufacturer’s instructions, and stored at -20°C prior

to further analysis. The quantity and quality of

extracted DNAs were measured using a NanoDrop

NC2000 spectrophotometer (Thermo Fisher

Scientiﬁc, Waltham, MA, USA) and agarose gel

electrophoresis, respectively.

The extracted DNA was amplified with two-step

PCR, with sample-specific 16-bp barcodes were

incorporated into the forward and reverse primers for

multiplex sequencing in the second PCR step. A total

of PCR amplicons were puriﬁed with Agencourt

AMPure Beads (Beckman Coulter, Indianapolis, IN)

and quantiﬁed using the PicoGreen dsDNA Assay Kit

(Invitrogen, Carlsbad, CA, USA). After the

individual quantification step, amplicons were pooled

in equal amounts, and Single Molecule Real Time

(SMRT) sequencing technology was performed using

the PacBio Sequel platform at Shanghai Personal

Biotechnology Co., Ltd (Shanghai, China).

3 RESULTS AND DISCUSSION

3.1 Microbial Alpha Diversity Analysis

The statistical results of the samples were shown in

Table 2. It showed Chao1 of MPSR was the largest,

followed by R and SBR, indicating that the

population abundance in MPSR is the highest. The

order of Shannon and Simpson value was R > MPSR

> SBR, indicating that the community diversity of

MPSR was better than SBR. The good's coverage

values of the three samples all reached 0.94, which

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

indicated that the high-throughput sequencing results

of the samples were in good agreement with the real

situation and could represent the real situation of the

samples.

Table 2: Statistics of activated sludge population abundance

and diversity index.

Index

Sample

R MPSR SBR

Chao1 458.43

484.48 418.42

Simpson 0.98

0.92

0.85

Shannon 7.20

6.02

4.91

Good's

coverage

0.94 0.93 0.94

3.2 Species Community Differences

The number of OTU clusters of R, MPSR and SBR

samples were represented by Venn diagram. As

shown in Figure 2, the OTU numbers of R, MPSR and

SBR were 387, 316 and 263 respectively, and the

species numbers of both reactors decreased after 30

days. The number of unique species in the three

samples was 159, 86 and 92, respectively, and owned

number of OTU was 79 (12.91%), which meant quite

different species communities. After the activated

sludge was cultured in their respective reactors, the

microbial environmental conditions changed, and

different dominant bacteria was formed in the system

due to the different internal structures of MPSR and

SBR.

Figure 2: Wayne diagram of OTU distribution of sludge

sample.

Species number and relative abundance were

counted from top20 phylum levels of the average

abundance of the sample. The results showed that

there were 2114, 3708 and 4712 species inside R,

MPSR and SBR, respectively. The number of species

in MPSR and SBR increased by 75.4% and 122.9%

respectively, indicating that the two processes

provided a good living environment for

microorganisms and improved species diversity.

Proteobacteia were major group in all the sludge

systems, followed by Bacteroidetes, Chloroflexi and

Actinobacteria. However, the abundance of sludge

samples varied significantly (Figure 3a).

3.3 Species Abundance Composition

As can be seen from Figure 3a, the dominant bacteria

phyla in SBR were Proteobactia (60.2%), Chloroflexi

(14.7%) and Bacteroidetes (11.9%), of which

Proteobactia was the most dominant phyla involved

in nitrogen and phosphorus removal and organic

matter degradation (Zhang 2015). The dominant

phyla of MPSR were Proteobacteia (29.7%),

Bacteroidetes (15.0%), Chloroflexi (7.6%),

Actinobacteria (6.9%), Firmicutes (3.4%) and other

phyla greater than 1% including Nitrospirae,

Plantomycetes and Verrucomicrobia, while another

32% of the species were not clear. Compared with

SBR, MPSR had more dominant bacteria phyla and

richer species.

As can be seen from Figure 3b, Thauera was the

main dominant bacteria of R, while its abundances

were reduced in both reactors, substitute for more

suitable for their own environment. Thiotrix occupied

a relatively high abundance in SBR. It belongs to

chemoautotrophic flora and plays a major role in

nitrification of the denitrification process. In MPSR,

nitrification and denitrification were in progress

simultaneously, and the dominant flora were various

functional flora such as Flavobacterium dominated by

heterotrophic denitrification and Thiotrix dominated

by nitrification (Zhang 2019).

(a) phylum

Study on Microbial Population Difference for the Treatment of Domestic Sewage between Micro-pressure Swirl Reactor (MPSR) and SBR

(b)genus

Figure 3: Species abundance of different sludge samples.

3.4 Species Composition Heat Map

In order to further compare the species composition

differences between samples, the abundance data of

top50 species in the average abundance were used to

draw a heat map for species composition analysis. In

the Figure 4, the red color block indicates that the

abundance of this species in this sample is higher than

that in other samples, and the blue color block

indicates that the abundance of this species in this

sample is lower than that in other samples.

The bacterial clustering results of three sludge

samples R, MPSR and SBR showed that there were

significant differences in microbial abundance

between the two reactors, which was due to the

effects of different circulating flow patterns and

oxygen environment on microbial flora. In MPSR

samples, the abundances of Propionicimonas

paludicola, Nitrospira sp. and Prevotella Copri were

larger, and in SBR samples, the dominant bacteria

were Thiothrix eikelboomii, Haliscomenobacter

hydrossis and etc.

Propionicimonas paludicola was a Gram-positive

bacterium, belonging to Actinobacteria. It was

facultative anaerobic and chemotrophic

heterotrophic, which could ferment and metabolize

glucose and other carbohydrates into a large amount

of acetic acid and propionic acid. Nitrospira sp. was a

gram-negative bacterium, belonging to Nitrospirae. It

was strictly aerobic and could oxidize nitrite into

nitrate to obtain energy which was the main nitrite

bacteria in the sewage treatment system (Siripong

2007). Prevotella Copri was a polymorphous

bacterium and did not produce spores. It was strictly

anaerobic and chemotrophic heterotrophic which

could use organic matter for anaerobic fermentation.

Therefore, compared with aerobic bacteria in SBR,

there were dominant microbial species of strict

anaerobic, strict aerobic and facultative anaerobic

inside MPSR. From the perspective of

microorganisms, the multiphase theory of MPSR was

proved and the function of simultaneous nitrification

and denitrification in multi-oxygen environment was

realized.

SBR R MPSR

Figure 4: Species level species composition heat map based on double clustering.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

4 CONCLUSIONS

Although water quality and intermittent operation

mode were the same, the two reactors had significant

differences in microbial community structure due to

different circulating flow patterns. Compared with

SBR, MPSR had more dominant bacteria phyla and

some functional bacteria of higher relative abundance

such as Flavobacterium and Thiotrix, and there

existed strictly anaerobic, strictly aerobic and

facultative anaerobic microbial species

simultaneously so as to produce higher species

diversity and population richness, which is accord

with the polyphase theory of the reactor.

ACKNOWLEDGEMENTS

The research was funded by the Project of science and

technology development plan of Jilin Province

(20210101079JC), Project of ecology and

environment department of Jilin Province (2021-14),

Project of science and technology fund of school

(320200030).

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Study on Microbial Population Difference for the Treatment of Domestic Sewage between Micro-pressure Swirl Reactor (MPSR) and SBR