Investigation of Microbial Diversity in Sludge Treatment Reed Bed

Junwen Ma

1,2 a

, Yubo Cui

1,3,* b

, Chengdong Ma

, Wanjun Zhang

, Zhongwei Zhang

and Ke Zhao

Key Laboratory of Biotechnology and Bioresources Utilization, Ministry of Education, Dalian Minzu University, Dalian

116600, China

School of Environment Science & Technology, Dalian University of Technology, Dalian, 116024, China

College of Environment and Resources, Dalian Minzu University, Dalian, 116600, China

Department of Marine Ecological Environment Information, National Marine Environmental Monitoring Center, Dalian,

116023, China

Key Laboratory of Songliao Aquatic Environment, Ministry of Education, Jilin Jianzhu University, Changchun, 130118,

China

Keywords: Sludge Treatment Reed Beds, Microbial Diversity.

Abstract: This paper fully characterized the diversity of the bacterial flora in sludge treatment reed beds (STRBs) by

sampling samples from the STRBs in stages and overall studying the changes in the bacterial flora in the

natural stabilization period, with sludge samples as the main focus, divided into bottom and surface sludge

discussions, and combined with reed samples and natural substrate samples to give auxiliary analysis. We

found that species changes of the dominant bacterial community in sludge mainly changed with different

periods of ecological stabilization, and the diversity as well as the homogeneity of the bacterial community

was increasing after the stabilization period, indicating that the root activity in plant growth can influence

the activity of nearby bacterial communities.

1 INTRODUCTION

At present, China's sewage treatment process is

becoming more and more mature, but the amount of

residual sludge generated by the sewage treatment

process is huge, so the disposal of residual sludge

should not be underestimated

(Wu, et al., 2000).

Specifically, the amount of residual sludge produced

today is up to more than 35 million tons, and its

water content is all around 80%. The main treatment

measures for residual sludge are composting,

incineration, landfill, drying and digestion, but still

80% of the sludge cannot be properly treated.

Therefore, the secondary pollution problem is still

prominent. Secondary pollution problems mainly

exist in terms of high sludge treatment costs, poor

https://orcid.org/0000-0001-5716-9446

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https://orcid.org/0000-0002-7481-0503

product marketing and the existence of pollution

transfer (Cui, et al., 2018). From a technical and

economic point of view, incineration and

composting for agricultural use are reliable

technologies for the treatment and disposal of huge

amounts of sludge, but the high investment and

operating costs hinder the widespread application of

incineration technology, and the high cost of

composting technology also requires consideration

of agricultural safety issues, which are the

challenges that must be faced in promoting the

technology. Therefore, sludge treatment or

stabilization requires consideration not only of

internal factors of operating technology, but also of

external factors such as economic costs and

environmental operation. The sludge treatment reed

bed (STRB) is a combination of traditional sludge

drying beds and artificial wetland technology, which

not only effectively dewater the remaining sludge,

but also additionally produce a mineralized

substance that can be used as a land improvement

and agricultural fertilizer

(Uggetti, et al., 2010,

Nielsen, et al., 2016, Hardej, et al., 2002). The

Ma, J., Cui, Y., Ma, C., Zhang, W., Zhang, Z. and Zhao, K.

Investigation of Microbial Diversity in Sludge Treatment Reed Bed.

DOI: 10.5220/0011180300003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 59-63

ISBN: 978-989-758-595-1

diversity of sludge flora in STRBs is important for

evaluating the effectiveness of STRBs in treating

residual sludge. Previously, there is still a gap in the

research on the diversity of bacterial flora in STRBs

during the stabilization period. With the continuous

advancement of technological innovations in

molecular biology, the use of denaturing gradient gel

electrophoresis (DGGE) to study microbial changes

in microecological environments has been widely

used, but a complete cycle study of microbial flora

diversity during the ecological stabilization of

residual sludge has not been carried out.

In conclusion, it is necessary and urgent to

investigate the changes of microbial flora in STRBs

during the stabilization period using DGGE.

2 MATERIALS AND METHODS

2.1 Sampling

The STRB system is divided equally into three units,

all of which are 3.0 m x 1.0 m x 1.3 m. Unit 1 is a

conventional STRB with an aeration structure (two

aeration risers); Unit 2 is a STRB with an aeration

structure (two aeration risers) and reeds are grown;

Unit 3 is a normal STRB with only reeds but no

aeration structure. The time period selected for the

study was eight months, from April to November in

the resting period, according to seasonal variations.

The reed bed is mainly composed of packing

layer and mud storage layer. The packing layer is

filled with 20 cm of slag, 20 cm of gravel, and 25

cm of British sand filter material in sequence from

bottom to top, of which 5 cm of coarse sand and the

rest are fine sand; the mud storage layer is set to 65

cm, In order to provide enough space for sludge

accumulation in the later stage. Aeration risers are

installed along 1/3 and 2/3 of the length of the drain

pipe and extend to the space above the mud storage.

2.2 Methods

In this experiment, DGGE was performed using Bio-

Rad's denaturing gradient gel system, and the basic

operating conditions for this experiment were

determined after repeated adjustments and tests. The

concentration of polyacrylamide gel denaturant used

in this study was 8%, and the gradient of denaturing

gel was 30-60% in 1× TAE buffer, and

electrophoresis was carried out at 18 mA for 18-20 h.

3 RESULTS AND DISCUSSION

3.1 Cluster Analysis and Diversity

Index of Bacteria

3.1.1 Analysis of May and November

Samples from Strbs

According to the results of cluster analysis, Figure 1

shows that lane 4 (5-2S) and lane 5 (5-3B) have the

lowest similarity with the rest of the samples,

indicating that the dominant bacteria in the bottom

sludge of unit 2, which is planted with reeds and

aerated, and the surface sludge of unit 3, which is

planted with reeds and not aerated, have similarity

and belong to two groups with the rest of the

samples during the budding stage of reed growth in

the natural stabilization period. In the natural

stabilization period of STRBs, the surface sludge

and bottom sludge of each unit did not belong to the

same taxon in the same sampling period. This can

also be seen by the greater differentiation between

11# (11-3B) and 12# (11-3S). In one unit, the

similarity between 1# (5-1B), 2# (5-1S), 7# (11-1B),

and 8# (11-1S) was not high. According to the

related study from the germination stage of reed, soil

respiration rate increased with the increase of

temperature, and soil temperature and near surface

temperature reached the highest value in July-

August, and soil respiration rate peaked

correspondingly in the vigorous growth period; after

entering the wilting stage, soil respiration in both

reed wetlands gradually decreased with the decrease

of soil temperature and near surface temperature,

due to the The lowest value of soil respiration rate

was reached in the overwintering period due to the

limitation of low temperature. Unit I, as a STRB, the

role of flora was mainly related to soil respiration

rate, indicating that the dominant flora of each

sample in Unit I were not in the same range. In unit

II, the dominant bacterial groups between 3# (5-1B)

and 10# (11-1S) belonged to the same taxon,

indicating that the sludge potential bacterial groups

in unit II planted with reeds did not change much

and had some stability.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

Figure 1: The dendrogram of the DGGE fingerprint of the V4-V5 gene of 16S rDNA of the bacteria from twelve sample. 1-

6: The samples in May; 7-12: The samples in November

The diversity values of the same sludge sample

in May and November were compared

longitudinally, and it was found that the difference

between the two units in the bottom sludge sample

was the smallest, and the H value increased from

1.44 (3#5-2D) to 1.61 (9#11 -2D); bed number 3

rose from 1.27 (5#5-3D) to 1.86 (11#11-3D), with

the largest fluctuation; while unit 1 dropped

significantly from 1.93 (1#5-1D) to 1.41 ( 7#11-1D),

in the process of research and application, the

diversity index represents the degree of species

diversity of each sample bacteria, and is a

comprehensive index of richness and uniformity.

Mud was closest to the degree of species diversity at

the beginning and end of the study cycle. The

surface sludge was analyzed, and it was found that

the fluctuation between the first unit and the third

unit was not large, and the second unit had a certain

fluctuation relatively. The H value increased from

1.40 (4#5-2B) to 1.72 (10#11-2D), The results

showed that in the comparison of the surface sludge

diversity index, the two units had the largest

difference in species diversity at the beginning and

end of the study period. It can be seen from Figure

3.6 that the fluctuation of the evenness index EH

value also characterizes the above trend. There is

little difference in the richness of the sample

colonies, the highest is 13 (11#11-3D), and the

lowest is 9 (5#5-3D).

On the whole, the diversity index in May and

November did not fluctuate much, and the sludge

had a certain degree of stability.

3.1.2 Analysis of July and September

samples from STRBs

According to the results of cluster analysis, Figure 2

shows that the reed samples (8#, 9#, 10#, 18#, 19#,

20#) and the sludge samples (from 1# to 7# and

from 11# to 17#) are clearly divided into two major

clusters with a similarity of 0.37. Comparing the

reed samples, it can be seen that the similarity

between the 9# (7-L2) and 10# (7-L3) samples is as

high as 0.94, indicating that these two samples of

reeds showed great consistency in July because they

were both growing in the sludge. In contrast, the

comparison of 19# (9-L2) and 20# (9-L3) revealed

that the reeds began to show some variability in

growth in September. the similarity between the reed

samples in July (8#, 9#, 10#) and September (18#,

19#, 20#) was arranged apart, indicating that the

reed endophytes' changed in July and September.

There are some obvious fluctuation patterns in

the sludge samples, within one unit, the samples 1#

(7-1B) and 2# (7-1S) in July do not belong to one

taxon and the similarity value between the two taxa

is 0.75, and the corresponding samples 11# (9-1B)

and 12# (7-1S) in September also do not belong to

one taxon and the similarity value becomes smaller

to 0.67, indicating that in the sludge with only

contemporaneous structure The difference in the

sludge surface and bottom layers of the sludge in the

dryer bed and the fluctuation of the dominant

bacterial group. A cross-sectional comparison of the

samples from the three units shows that the sludge

samples 5# (7-3B) and 6# (7-3S) from July were not

matched to the same taxon, but the samples 15# (9-

3B) and 16# (9-3S) from September had high

similarity values (0.85) and belonged to the same

taxon; a longitudinal comparison revealed that 5# (7-

3B) and 15# (9-3S) belonged to two taxa (0.71) and

6# (7-3B) and 16# (9-3S), indicating that the three

units (reedbeds without aeration structures) changed

significantly after the growth period. Among the

three units, the lanes of 3# (7-2B), 4# (7-2S) and 13#

(9-2B) were sequentially adjacent to each other in

the analysis chart with high similarity; 14# (9-2S)

represented a sludge sample at the peak of reed

Investigation of Microbial Diversity in Sludge Treatment Reed Bed

growth with some variability from the above

samples (similarity value 0.71). The natural

substrate samples 7# and 17# belong to one taxon,

but the similarity is low (0.59), indicating that the

natural substrate samples also changed in July and

September.

Figure 2: The dendrogram of the DGGE fingerprint of the V4-V5 gene of 16S rDNA of the bacteria from twenty sample.

1-10: The samples in July; 11-20: The samples in September.

Compared with the diversity index in July and

September, it was found that the fluctuation of the

diversity index of the sludge sample was

significantly larger than that of the reed sample, and

the diversity index of the natural sediment sample

was closer to the reed sample, which was relatively

stable. The diversity index of the reed samples was

similar in two months. Compared with the

uniformity index, it was found that the reed samples

in the second unit had the most stable changes

before and after (9#, 19#).

In the control of sludge samples, the one-unit

bottom sludge diversity index without reed plants

fluctuated the least, and the H value changed from

1.40 (1#7-1D) to 1.44 (11#9-1D). The bottom sludge

diversity index of the three units planted with reeds

fluctuated the most, and the H value changed from

2.03 (5#7-3D) to 1.20 (15#9-3D). In the comparison

of the surface sludge, the diversity index of pool 1

dropped significantly, and the diversity index of

pool 2 fluctuated less. In general, the diversity index

of samples in September was lower than that of

samples in July.

The trend graph of the evenness index EH value

also characterizes the above trend. There is a certain

difference in the abundance of sludge sample

colonies, the highest is 14 (5#7-3D), the lowest is 6

(16#9-3B), and the richness of natural sediment is 5

(7#7-T and 17#9-T); the richness of the reed

samples differed little, both in July and in

September.

3.2 Bacterial Species and Affinity

Analysis

3.2.1 Species and Affinity Analysis of May

and November Samples

The research showed that it can be concluded that

each band represents Bacteria (bacteria), bands 2, 5

and 6 do not specify the specific dominant bacterial

genus, and after comparison, it is known to belong to

the environmental sample species.

Band 8 belongs to Bacteroidetes (Bacteroidetes),

which is widely found in nature, including soil,

sediment and seawater, as well as animal skin and

viscera, and according to the results, this species is

the dominant species in the samples of each unit in

May, while the dominant characteristics in the

samples of November are concentrated in the bottom

sludge.

The rest of the bands in the map represent the

dominant genus of bacteria belong to Proteobacteria

(Phylum Amoebae), bands 3, 4, 7, 11, 12, 13 and 14

are from Gammaproteobacteria (γ-Amoebacteria),

bands 4, 13 and 14 are bacteria Rhodanobacter, band

3 is bacteria Dyella, bands 8 and 12 are only

compared to Xanthomonadaceae (Order

Xanthomonadaceae).

Bands 9 and 10 both belong to

Betaproteobacteria (β-Amastigotes), band 9

represents Burkholderiales (Burkholderiales) -

Comamonadaceae (Trichomonadaceae) -

Simplicispira; band 10 represents Hydrogenophilales

(Hydrogenophilales) - Hydrogenophilaceae

(Hydrogenophilaceae) - Thiobacillus (Thiobacillus

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

spp.), a dominant bacterium that grows mainly in

environments with pH values between 3 and 4 and

produces sulfuric acid to improve fertilization

efficiency.

3.2.2 Species and Affinity Analysis of July

and September Samples

The comparison results showed that band 4 was

Uncultured Acidobacteriales, which belongs to

Acidobacteria, a newly discovered bacterium and

less studied.

Band 6 was Uncultured Sphingobacteria, i.e.

Sphingobacteria (Phylum Sphingobacteria), which

are more frequent in the dominant group analysis

and have been shown to remove ammonia nitrogen

from water. This band only appeared in the sludge

samples of the second unit in July and in the bottom

sludge of the first unit in the same period, indicating

that the sludge of the second unit in July had a

certain bacterial richness.

Band 7 is Uncultured Bacteroidetes, which is

Bacteroidetes (phylum Bacteroidetes), a specialized

anaerobic bacterium that generally exists widely in

manure wastewater and thickened sludge. The

presence of this band in lanes 2# (7-1S) and 5# (7-

3B) indicates that the sludge in units 1 and 3 showed

an anaerobic environment in July, and presumably

the sludge in unit 2 had a higher oxygen content.

Band 10 was Gloeobacter, which showed

predominance in all samples. It is a Cyanobacteria

(phylum Cyanobacteria), a group of bacteria that

produces oxygen. It belongs to Gloeobacter (genus

Gloeobacter) in the Gloeobacteraceae (family

Mucoraceae).

Band 16 is Uncultured Alcanivorax, also

belonging to Gammaproteobacteria (γ-Amastigotes)

in Proteobacteria (Phylum Proteobacteria), but

Alcanivorax (Alcanivorax spp.) in Oceanospirillales

(Order Oceanospirillales). The profile showed the

occurrence of this class of bacteria in the second unit

of sludge as well as in natural sludge in July.

4 CONCLUSIONS

In this paper, we found significant differences in the

DGGE profiles of bacterial populations in different

periods and sampling locations by sampling samples

in STRBs. The changes in the dominant bacterial

species in sludge mainly changed with different

periods of ecological stabilization, and the diversity

as well as the homogeneity of the dominant bacterial

flora generally increased after the stabilization

period of action, indicating that the root activity in

plant growth can stimulate the activity of nearby

bacterial flora to some extent.

Sequence comparison revealed that during the

stabilization period of STRBs, the dominant bacteria

were Bacteroidetes and Proteobacteria at lower

temperatures, and Thiobacillus (Thiobacillus spp.),

which can produce sulfuric acid to improve

fertilization efficiency, was always present. The

dominant species were widely distributed at higher

temperatures, including Proteobacteria (Phylum

Anamorphobacteria), Acidobacteria (Phylum

Acidobacteria), Sphingobacteria (Phylum

Sphingobacteria), Bacteroidetes (Phylum

Anamorphobacteria), and Cyanobacteria (Phylum

Cyanobacteria). Among them, Acidobacteriales

(Acidobacteria), which is abundant in soil, appeared

in all samples of sludge and natural substrate.

Gloeobacter which can produce oxygen was shown

in all samples at high temperature. The specialized

anaerobic bacteria, Bacteroidetes (phylum

Sphingobacter), was not shown in the rest of the

samples, except in the samples of 1S and 3B in May

and July.

ACKNOWLEDGEMENTS

The research was financed by the Natural Science

Foundation of Liaoning, China (2020-MZLH-02)

and Science and Technology Innovation Foundation

of Dalian, China (2018J12SN080).

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Investigation of Microbial Diversity in Sludge Treatment Reed Bed