Accepted Manuscript

Microbial community evolution of black and stinking rivers during in situ re-
mediation through micro-nano bubble and submerged resin floating bed tech-
nology

Yanmei Sun, Shiwei Wang, Junfeng Niu

PII: S0960-8524(18)30346-8
DOI: https://doi.org/10.1016/j.biortech.2018.03.008
Reference: BITE 19647

To appear in: Bioresource Technology

Received Date: 27 January 2018

Revised Date: 28 February 2018
Accepted Date: 1 March 2018

Please cite this article as: Sun, Y., Wang, S., Niu, J., Microbial community evolution of black and stinking rivers
during in situ remediation through micro-nano bubble and submerged resin floating bed technology, Bioresource
Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.03.008

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 Microbial community evolution of black and stinking rivers during in situ

remediation through micro-nano bubble and submerged resin floating bed

technology

Yanmei Suna,†, Shiwei Wangb,†, Junfeng Niua,*

a
Research Center for Eco-Environmental Engineering, Dongguan University of

Technology, Dongguan, Guangdong 523808, P.R. China

b
School of Chemical Engineering and Energy Technology, Dongguan University of

Technology, Dongguan, Guangdong 523808, P.R. China

†
Equal contributions

* Corresponding author. Tel./Fax: +86-769-39018706

E-mail address: niujf@dgut.edu.cn

1
ABSTRACT

Microbes play important roles during river remediation and the interaction

mechanism illustration between microorganisms and sewage is of great significance

to improve restoration technology. In this study, micro-nano bubble and submerged

resin floating bed composite technology (MBSR) was firstly used to restore two black

and stinking urban rivers. After restoration, the water pollution indices such as

dissolved oxygen (DO), ammonia nitrogen (NH4+-N), total phosphorous (TP),

chemical oxygen demand (CODCr), water clarity, and the number of facial coliform

were significantly improved. Microbial community composition and relative

abundance both varied and more aerobic microbes emerged after remediation. The

microbial changes showed correlation with DO, NH4+-N, TP and CODCr of the rivers.

In summary, the MBSR treatment improved the physiochemical properties of the two

black and stinking urban rivers probably through oxygen enrichment of micro-nano

bubble and adsorption of submerged resin floating bed, which thereby stimulated

functional microbes to degrade pollutants.

Keywords: Black-stinking river; Micro-nano bubble; Submerged resin floating bed;

Microbial community evolution

2
1. Introduction

In recent years, agricultural and industrial development and urbanization has

impaired river health in China. The rivers especially flowing through cities were

frequently contaminated by various anthropogenic pollutants such as domestic sewage,

industrial wastewater, solid waste and so on, and the number and severity of polluted

rivers both were increasing year by year in China (Noorhosseini et al., 2015; Yuan et

al., 2012). The heavily polluted rivers exhibit seasonal or perennial black and stinking

phenomenon, which results in loss of aquatic ecosystem. The deterioration of the

water quality adversely affects people life and poses a serious threat to human health

(Viswanathan and Schirmer, 2015). The black and stinking urban rivers have become

one of the important water environment issues and aroused widespread concern.

Recently, a great deal of financial and material resources was invested to control or

restore water pollution in China. In general, the physiochemical and biological

methods such as aeration, aquatic plants replantation, bacterial inoculation and

enzyme technology were used to eliminate pollutants from rivers (Haque and Kwon,

2017; Liu et al., 2016; Mi et al., 2015). These measures may be effective to remediate

light eutrophic rivers with mental or organic pollutants but not for black and stinking

urban rivers, which contain high concentration of pollutants and low dissolved oxygen

(DO). Therefore, the target repair technology is urgent to be developed for black and

stinking urban river harnessing.

Black and stinking phenomenon of urban rivers is mainly caused by organic and

inorganic pollutants with extremely high levels (Ji et al., 2017). Excessive pollutants

3
discharged into rivers, a part of them precipitated in sediment to form endogenous

pollution, and the others were degraded by aerobic microorganisms. The degradation

of pollutants consumed DO of rivers, which caused hypoxia of water and enormous

multiplication of anaerobic microbes. The anaerobic microorganisms decompose

organic matter to produce a large amount of foul gas such as H2S, NH3 and volatile

organic sulphides and at the same time to form some black substances such as FeS,

MnS and so on. Finally, the water body shows strong reducibility, pan-black and stink

smell. The ecological structure of the black and stinking urban rivers is out of balance

and thereby they are not suitable for aquatic life due to loss of the ecological function.

Thus, how to increase DO of water body to stimulate aerobic microorganism to

reduce high-load pollutants is one key factor in the remediation process of black and

stinking urban rivers.

Under these circumstances the micro-nano bubble and submerged resin floating

bed composite technology was applied to restore two actual black and stinking urban

rivers. The micro-nano bubble technology has been reported to use in water treatment

(Agarwal et al., 2011; Hu and Xia, 2017). Compared to ordinary aeration, the

micro-nano bubble technology can produce smaller bubbles with diameters of several

tens of micrometers and nanometers, which have slow rising speed and high

self-pressurization and gas dissolution capability (Agarwal et al., 2011; Hu and Xia,

2017). In contrast, as far as we know, few studies have been reported to investigate

the effect of submerged resin floating bed technology on remediation of black and

stinking urban rivers. In order to increase specific surface area, in the submerged resin

4
floating bed system the polymer resins were designed to be less than 1 mm by

electrostatic spinning method and it was expected to absorb more suspended particles

and contaminants. In this study, the composite technology of micro-nano bubble and

submerged resin floating bed (MBSR) was firstly used to restore two actual black and

stinking rivers.

It is known that free-living microorganisms, such as planktonic bacteria, play an

important role in biogeochemical cycling of watercourse. Microbes have been

demonstrated to decompose, transform and mineralize nutrients, organic matter and

recalcitrant contaminants in urban rivers (Beale et al., 2017; Chen et al., 2017; Pi et al.,

2017). Microbial communities interacted with pollution sources and they showed high

sensitivity towards the altered environmental status (Abram et al., 2014; Lee et al.,

2017; Zeglin et al., 2011). Therefore, analyzing the impact of MBSR on microbial

population would help to illustrate the restoration mechanism of the black and

stinking urban rivers by the technology.

In this study, two black and stinking rivers were chosen to evaluate the effect of

MBSR on restoration of severely polluted water body in the south of China. To

demonstrate the remediation mechanism of MBSR, microbial community dynamic

evolution, the correlation between microbial community and pollutants removal, and

the absorption effect of MBSR on suspended pollutant in water body were

investigated. This study provided more understanding about the ecological effects of

MBSR on stimulating in situ microorganisms to remove contaminants and improve

the biological self-cleaning capacity of black and stinking rivers.

5
2. Materials and methods

2.1. Sampling sites

The two polluted urban rivers (designed as River A (N22°57′33″, E113°45′45″)

and River B (N22°47′45″, E113°46′55″)) of this study flow through urbanized region

of the southern China. The full length of River A was 462 m, and the sewage drained

directly into River A through municipal sewage mixing system and then flowed into

neighboring reservoir. River B has an 1854 m open-channel and an 1458 m

closed-channel. The water quality of River B was affected by tidal water of the

adjacent river and displayed some fluctuation every day. The flow rates of River A

and B were 0.2~0.3 m/s and ~0.1 m/s, respectively, and the temperature was both 25

± 2°C for the two rivers. The surrounding environment of the two rivers was

complicated and there are a lot of residents and factories. The two rivers were

subjected to a high level of domestic, agricultural and industrial contamination

throughout its course for a long time, which caused serious water quality deterioration.

The water body of the two rivers were mild black and stinking status before

restoration.

The micro-nano bubble and submerged resin floating bed system were combined

to restore the two polluted rivers. During the river treatment process, three areas in the

upper, middle and down stream of River A and five sites of two channels of River B

were monitored. In the twenty days before and after restoration, the water samples

were taken every day for water quality analyses. To monitor the microbial response to

MBSR treatment, some water samples were retrieved on the 3rd and 7th day of

6
restoration. Five hundred milliliters of each water sample was filtered through 0.22

µm membrane (Millipore, Billerica, MA, USA) and then stored in -80 °C for DNA

extraction.

2.2 Physicochemical and bacteriological analyses

pH, DO, oxidation reduction potential (ORP) and conductivity of water samples

were measured on-site by a Hach HQ40d multi-probe meter (Hach, Loveland, CO,

USA). The chemical oxygen demand (CODcr), ammonia nitrogen (NH4+-N), total

nitrogen (TN) and total phosphorous (TP) were examined according to the standard

method (APHA, 2012) and biochemical oxygen demand (BOD) was measured by

using Hach’s method 8000 (Hach DR/890 colorimeter procedures manual) (Morita et

al., 2011). The number of Escherichia was counted by filtering different dilutions of

water samples and then incubated the filters on Chromocult coliform agar (Merck,

Darmstadt, Germany) at 37°C for 24 h.

2.3 Genomic DNA extraction and high-throughput 16S rRNA sequencing

Microbial genomic DNA was extracted using the PowerWater® Sterivex™ DNA

Isolation Kit following manufacturer’s instructions (Mo Bio Laboratories, Carlsbad,

CA, USA). The extracted DNA samples were amplified with a set of primers targeting

the hypervariable V3-V4 region of the 16S rRNA gene (the sequences of 341F/805R

primer set were CCTACGGGNGGCWGCAG and GACTACHVGGGTATCTAATCC,

7
respectively) (Herlemann et al., 2011). The concentration of purified amplicons from

each sample was measured using a Qubit dsDNA HS Assay Kit (Life Technologies,

Eugene, OR, USA). Amplicons were processed using Nextera XT DNA Library

Preparation Kit according to Illumina’s protocol. High throughput sequencing was

performed on an Illumina MiSeq platform by Sangon Biotech (Shanghai, China).

2.4 Data analysis for microbial community composition

The raw paired-end reads were assembled and then quality was filtered using

QIIME v1.9.0. After trimming adapt sequences of the raw reads, the high-quality

reads were clustered into operational taxonomic units (OTUs) with 97% sequence

identities. The RDP classifier was used to annotate taxonomic information for each

OTU representative sequence. To analyze the diversity at the alpha level, Chao1 and

ACE indices were computed to estimate microbial richness, and Shannon and

Simpson indices were used to estimate microbial diversity. The common and unique

OTU numbers among samples were analyzed by using the R VennDiagram package.

Hierarchical cluster analysis was carried out using Bray–Curtis similarity based on the

abundance of all OTUs in R software with the statistical package vegan. Principal

coordinate analysis (PCoA) was performed to analyze the microbial community

dissimilarity using the R statistical vegan package. The relationship between water

quality condition and microbial community was explained by Mantel test and

canonical correlation analysis (CCA), which were calculated by the R ade4 and vegan

packages, respectively. Circos and Heatmap were used to visualize the dominant

8
microbes before and after restoration, which were performed in R software with

circlize and pheatmap package, respectively. The significant representative

microorganisms of each sample were distinguished by LefSe analysis. The microbial

function was predicted by Clusters of Orthologous Group (COG).

3. Results and Discussion

3.1 Water quality improvement in the two black and stinking rivers

Some water quality indices, such as DO, pH, conductivity and so on, were used

for river classification and urban impact assessment (Kannel et al., 2007). Analysis of

the physicochemical parameters revealed that the two urban rivers were both close to

mild black and stinking water body (Fig. 1). Before restoration the DO values of

detection sites in Rivers A and B were 1.10–4.53 mg/L and 1.5–5.47 mg/L,

respectively, and it was < 0.1 mg/L for part of locations. After MBSR treatment the

public sense perception of water body improved obviously. The aeration system

producing micro-nano bubble significantly increased the DO values of two rivers (Fig.

1A). In addition, ORP has a three-fold increase in the two rivers (Fig. 1A). The rivers’

pH was about 7.0 and it wasn’t affected by MBSR treatment. In contrast, the

conductivity remarkably reduced in River A, indicating the effect of MBSR on

reducing the concentration of inorganic salt and heavy metal in urban rivers.

In this study, the DO increase level of the two urban rivers by micro-nano bubble

was similar with previous studies, which were achieved by intermittent aeration or

aeration combined with plants (Gu et al., 2015; Zhu et al., 2017). However, most of

9
them were lab or pilot mesocosm experiments (Li et al., 2014). The ORP value was

related to DO of urban rivers and high ORP was found to alleviate ammonia and

phosphorus release in the sediment and then to reduce river eutrophication (Li et al.,

2016). In previous study, metals such as chromium, copper and nickel were reported

to interact with organic matter in the aqueous phase and then settled in the sediment to

cause pollution (Pardo et al., 1990). Micro-nano bubble hinders sedimentation and it

was presumed that the decrease of conductivity was mainly due to the adsorption

effect of the submerged resin floating bed system. Metal and salt ions might combine

with soil or other solid-contaminating particles and they were easy to attach onto the

submerged resin floating bed system along with micro-nano bubble flow. The

scanning electron microscope result confirmed that the submerged resin floating bed

system absorbed abundant particles, crystals and bacteria cells. In summary, DO and

ORP were significantly increased and conductivity was obviously decreased by

MBSR treatment, which was profitable for aerobic bioremediation to remove organic

pollutants and heavy metals.

Excessive organic matter is the main cause of black and stinking urban rivers,

which is reflected by the values of BOD and COD in water body. The value of COD

was four times of that of BOD in the two rivers, suggesting there were a lot of organic

contaminants that could not be degraded by microbes in the rivers (Fig. 1B). After

remediation COD of River A and B decreased 41.49% and 36.37%, respectively. New

pollutant analysis of the resin adsorbents showed that seventeen medicines and

antibiotics such as climbazole, sulpiride, diphenhydramine, ofloxacin and

10
azithromycin were present, consistent with the absorption effect of the submerged

resin floating bed system on pollutants in the two rivers. The previous study also has

demonstrated that resin could adsorb organic compound in solution by the

hydrophobic part (Zhao et al., 2013).

Point and non-point pollution of nitrogen and phosphorous enter water body and

lead to eutrophication of urban rivers. In River A, the concentrations of NH4+-N and

TP decreased from 7.68 to 4.51 mg/L and from 1.13 to 0.73 mg/L, respectively (Fig.

1C). In contrast, MBSR treatment influenced the NH4+-N and TP contents of River B

little. River B was replenished mainly through tidal water of the adjacent river that

was heavily polluted, and thereby the removal effect of NH4+-N and TP were not as

obvious as River A. Water clarity was increased from 28.1 to 58.4 cm for River A and

from 18.1 to 34.1 cm for River B, respectively (Fig. 1D). The number of facial

coliform was remarkably decreased in the two rivers (Fig. 1D), which could be

attributed to the absorption of the submerged resin floating bed system and nutrition

decrease of the two rivers. Taken together, MBSR showed good effect on the

remediation of the two urban rivers with completely different characteristics. The

black and stinking phenomenon was eliminated in a short period. The black water

body turned green and heavily stinking smell disappeared.

3.2 Effect of MBSR treatment on the urban rivers’ microbial profile

River microbes respond to environmental changes such as physicochemical

conditions and organic and inorganic pollutant levels. In this study, the sequencing

11
results showed that the number of clean sequences was from 32229 to 62129 and the

coverage was more than 97.9%, which reflected the real microbial composition in the

rivers. Before restoration, there were 1621 OTUs and 1858 OTUs in River A and B,

respectively. The Chao and ACE results showed the microbial community richness

was significantly decreased in the two rivers (p < 0.05) and ~50% OTUs disappeared

after MBSR treatment. However, Shannon and Simpon indices displayed that the

diversity changed little, indicating the microbial evenness was higher in the repair

process of the rivers. Further analysis revealed that 391 to 766 unique OTUs were

detected in the restored rivers, indicating the restoration treatment might stimulate

some microbes, which were present but not metabolically active in the two mild black

and stinking rivers. Compared to common and unique genes, the lower core OTU

numbers demonstrated obvious variation of microbial community in the initial stage

of the river restoration. Microbes had high sensitivity to changing environment such

as physiochemical conditions and concentration of nutrition and contaminants in

rivers (Zeglin et al., 2015). During the river restoration process, the submerged resin

floating bed system absorbed the microorganisms attached onto solid pollutants, and

on the other hand, the decreased values of COD and BOD indicated lower nutrition

for microbial growth in the rivers. Thus, the microbial richness and diversity

significantly reduced after restoration. However, some of the microbial lineages that

were metabolically inactive in the black and stinking urban rivers might be stimulated

in the process of MBSR remediation.

In order to illustrate the microbial community evolution before and after the river

12
restoration, PCoA and the cluster tree based on Bray-Crutis were constructed

according to the OTU distribution. PCoA1 and PCoA2 explained 23.79% and 19.45%,

respectively (Fig. 2). For each river, the restored samples were grouped together and

were separated from the unrepaired samples (Fig. 2). The cluster tree also showed

similar results, suggesting MBSR treatment has important effects on microbial

community composition. Besides, the microbial community of A0 (untreated River A)

and B3 (the 3th day of River B restoration) was clustered together, suggesting there

were some common microbes in the two stages of the two rivers. The initial pollutant

level of River B was higher than that of River A (Fig. 1), and the pollutant levels of

A0 and B3 might be similar, indicating the microbial community was related to the

polluted level of the rivers. In addition, with the extension of repair time the microbial

community of B7 was clustered with A3 and A7, again indicating that the microbial

community changed with the gradually improved water quality of the two rivers. In

the study on microbial community of sediment, the bacterial community was clustered

according to nitrogen pollution concentration (Li et al., 2016). Another study

suggested that the bacterial biomass varied with the distance of sewage discharge

point and it was related with the COD level of the river (Lin et al., 2015). It has been

reported that microbial community responsible fore aromatic biodegradation was

acclimated after the oil spill in the river (Acostagonzález et al., 2013). In all, the

results indicated that microbes were sensitive to adjust their composition according to

the polluted condition of rivers.

13
3.3 Microorganisms’ variation after MBSR remediation

Microbial community composition is a major driving factor of biogeochemistry

and energy cycling and is also a pollution indicator of urban river. To identify which

microbial groups altered in the process of river restoration, the dominant microbes

were analyzed at different classification levels. Gammaproteobacteria and

Flavobacteriia both were predominant in the two unrestored urban rivers (Fig. 3).

Besides, Betaproteobacteria was also dominant in River A and Alphaproteobacteria

was the main group in River B. After restoration, the above microbial groups were

still dominant, but the relative abundance changed. The relative abundance of

Alphaproteobacteria, Sphingobacteriia, Cytophagia and Actinobacteria increased with

the extension of restoration period (Fig. 3). Alphaproteobacteria are widely distributed

marine plankton that constitute over 10% of the open ocean microbial community,

Actinobacteria, Cytophagia and Flavobacteria clusters were found in oligotrophic

freshwater, and these planktonic microbial members were reported to contribute to

biopolymer degradation such as polysaccharide and protein (Sack et al., 2014).

Actinobacteria, terrestrial or aquatic, belongs to a phylum of Gram-positive bacteria

and they behave much like fungi, helping to decompose organic matter.

Actinobacteria has been reported to be significantly higher in all the sediment samples

than that in the surface water (Ibekwe et al., 2016), suggesting MBSR treatment might

promote the migration of microorganisms from the sediment into the overlying water.

Taken together, the results demonstrated that MBSR treatment might activate some

microbial groups related to the degradation of organic matter in the restoration

14
process of the two urban rivers.

Further analysis displayed that Acinetobacter, Acidovorax, Novosphingobium

and Cloacibacterium were abundant before and after restoration in the rivers (Fig. 4).

The MBSR treatment caused the variation of dominant genus. For example, some

microbes such as Tolumonas, Bacteroides, Escherichia, Azonexus and

Macellibacteroides were abundant in the polluted rivers, but their content was lower

after restoration. Tolumonas sp. was found to be present in anoxic or aerobic

freshwater lake sediment or activated sludge, which produced electricity or toluene

(Caldwell et al., 2011; Luo et al., 2013). Macellibacteroides sp. was reported to be

isolated from an up flow anaerobic filter treating abattoir wastewaters (Jabari et al.,

2012). The above results suggested the microorganisms survived under the low DO

condition were prevailed in the two polluted rivers before restoration. In addition,

Bacteroides and Escherichia species were dominant in the two rivers, which normally

make up the most substantial portion of mammalian gastrointestinal flora. The colony

count result of facial coliform showed these microbes were abundant, indicating

serious pollution from domestic water in the two rivers.

In contrast, the improved water quality was profitable for the growth of

Sediminibacterium, Cellvibrio, Rheinheimera, Limnobacter and Pseudarcicella, and

after restoration their abundance was higher than before restoration (Fig. 4). Most of

these genera were Gram-negative, strictly aerobic and motile bacteria such as

Sediminibacterium and Rheinheimera, which were reported to be predominant in

freshwater, seawater and sediment (Baek and Jeon, 2014; Kang et al., 2014; Qu and

15
Yuan, 2008). Besides, some of them were functional microbes. For example,

Cellvibrio can degrade cellulose and Limnobacter species have the ability to oxidize

thiosulfate (Berg et al., 2010). The results indicated that micro-nano bubble might

stimulate aerobic bacteria and their metabolic activity favored the removal of

contaminants. In addition, the adsorption of the submerged resin floating bed system

caused the microbial community variations in the rivers. The predominant isolates

from the submerged resin floating bed system were identified to be Acinetobacter,

Aeromonas and E. coli, which confirmed that the resin absorbed fecal coliform and

thereby decreased the microbial abundance of the rivers. Thus, MBSR is favorable to

the removal of fecal coliform and pollutants probably by stimulating the growth of

aerobic microorganisms in the two black and stinking urban rivers.

The significant representative microbial community was found in different

restoration phases of polluted rivers. For example, Escherichia, Shigella, Zoogloea

and comamonas were abundant in A0, Chromatiales, Terracoccus and Azospira were

enriched in A3, and Neisseriales and vogesella were predominant in A7. This

suggested that microbial community was highly sensitive to surrounding environment

and they adjusted the community composition according to different sources of

pollutants such as oil spills and domestic and industrial wastewater (Wang et al., 2017;

Xie et al., 2016; Zeglin et al., 2015).

3.4 Microbial function during the process of MBSR treatment

The function of microbial community in river ecosystem played important roles

16
during pollution removal. To get insight into biological processes involving in the

microbial community before and after restoration, the microbial functions were

predicted according to COG. The COG categories related to J (translation, ribosomal

structure, and biogenesis), K (transcription) and E (amino acid transport and

metabolism) were more abundant before restoration in River A. In contrast, microbes

responsible fore secondary metabolites, transport and catabolism were enriched after

restoration in River A (Fig. 5).

The black and stinking urban rivers with high COD, BOD and NH4+-N were

eutrophic, which were favorable to the growth and reproduction of microorganisms,

and the microbial functional categories responsible for transcription and translation

were abundant. This was consistent with the results of the high microbial abundance

and the large number of coliforms before restoration. In addition, the secondary

metabolism of microbial community was increased after restoration in River A.

Microbial secondary metabolites provide numerous pharmaceutical agents such as

antibiotics and immunosuppressive compounds, and thereby inhibit the growth of

other microorganisms (Coleman et al., 2011). This might be the response of

microorganisms to nutrient reduction in rivers where different microorganisms

competed for vegetative growth. In contrast, due to the impact of the adjacent polluted

tide river, the nutrient removal efficiency of River B was lower than that of River A.

Therefore, the corresponding microbial function did not show significant change in

River B. The changes of microbial community function indirectly reflected the effect

of MBSR treatment on nutrient removal of the two black and stinking urban rivers.

17
3.5 Correlation analysis between physiochemical characteristics and microbial

community

The observation of significantly improved water quality and varied dominant

genera provoked us to examine possible linkage between microbial community

functional structure and physiochemical parameters of the two rivers. To discern the

major environmental variables correlated with microbial community, CCA was

carried out. Four variables (DO, COD, TP and NH4+-N) were selected on the basis of

variance inflation factor (VIF), which formed a significant CCA model (P < 0.05).

Two axes explained 65.4% of microbial community variation (Fig. 6), indicating that

these four variables were major factors shaping microbial community. This finding

was further verified by Mantel test as DO, ORP, COD, BOD, TP and WC showed

correlation with microbial functional structure in River A (P < 0.05), but no separate

variables demonstrated significant correlation with microbial community in River B

(Table 1).

In the CCA biplot, DO appeared to be a major factor affecting the microbial

functional structure in River A (Fig. 6). The importance of DO for microbial activity

in water has been reported in previous studies (Simon et al., 2014; Vanderzalm et al.,

2013). In addition, NH4+-N, TP and COD were vital for microbial community

functional structure of River B. The high concentrations of pollutants and microbes in

adjacent polluted rivers flooded into river B with tidal water, which affected microbial

functional structure as well as the removal of pollutants. Therefore, physiochemical

18
factors together with the pollution level of the urban rivers affected the microbial

community of River B. The Linkage between microbial functional potential and

wastewater constituent was also revealed in the process of municipal wastewater

treatment (Sun et al., 2016). In summary, MBSR adjusted the physical and chemical

conditions of the two urban rivers mainly by changing DO, redox potential and

adsorption, shaped the composition of microbial community, and thereby

stimulated aerobic microbial functional groups to promote the degradation of

pollutants and improvement of water quality.

4. Conclusion

MBSR was applied to remediate two black and stinking urban rivers. After

MBSR treatment, the water quality was improved a lot, suggesting MBSR can be

used for in situ remediation of polluted rivers in future. Microbial richness, especially

the number of facial coliform, was significantly decreased in the rivers. The relative

abundance of the predominant phyla varied with physiochemical conditions, and

some aerobic microorganisms responsible for pollutant degradation were stimulated

in the process of MBSR remediation. Thus, MBSR improved water quality of polluted

rivers probably through the synergistic effects of physiochemical and biological

reaction.

Acknowledgments

This work was supported by the National Science Fund for Distinguished Young

19
Scholars (No. 51625801), the Scientific Research Foundation for High-level Talents

and Innovation Team in Dongguan University of Technology (No.

KCYKYQD2016001), the Guangdong Innovation Team Project for Colleges and

Universities (No. 2016KCXTD023), and the Guangxi innovation-driven

development project, Research and development of key technologies for pollution

control and quality improvement of water environment in malodorous black river (No.

AA17202032).

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Figure captions

Fig. 1. The water quality improvement of the two black and stinking rivers after

MBSR restoration (A: DO and ORP; B: COD and BOD; C: NH4+-N and TP; D: WC

and FC).

Fig. 2. PCoA analysis demonstrated the dissimilarity of microbial community

composition in the different treatments.

Fig. 3. Relationship between the samples and microbes at the class level was

displayed by the Circos analysis, showing the dominant microbial composition in the

different restoration phases of the two black and stinking urban rivers.

Fig. 4. The response of the predominant genera to MBSR restoration in the two black

and stinking urban rivers.

Fig. 5. Functional groups according to COG categories (clusters of orthologous

proteins) displayed the difference of microbial function before and after restoration in

River A (The left panel showed the abundance ratio of different functional groups

before and after restoration; the middle showed the percentage of functional group

abundance within the 95% confidence interval; the rightmost value is p value; p <

0.05, indicating significant difference).

Fig. 6. Canonical correspondence analysis (CCA) of microbial community and

physiochemical properties in the two urban rivers.

27
Fig. 1. The water quality improvement of the two black and stinking rivers after

MBSR restoration (A: DO and ORP; B: COD and BOD; C: NH4+-N and TP; D: WC

and FC).

28
Fig. 2. PCoA analysis demonstrated the dissimilarity of microbial community

composition in the different treatments.

29
Fig. 3. Relationship between the samples and microbes at the class level was

displayed by the Circos analysis, showing the dominant microbial composition in the

different restoration phases of the two black and stinking urban rivers.

30
Fig. 4. The response of the predominant genera to MBSR restoration in the two black

and stinking urban rivers.

31
Fig. 5. Functional groups according to COG categories (clusters of orthologous

proteins) displayed the difference of microbial function before and after restoration in

River A (The left panel showed the abundance ratio of different functional groups

before and after restoration; the middle showed the percentage of functional group

abundance within the 95% confidence interval; the rightmost value is p value; p <

0.05, indicating significant difference).

32
Fig. 6. Canonical correspondence analysis (CCA) of microbial community and

physiochemical properties in the two urban rivers.

33
Table 1

Relationship between microbial community and physiochemical characteristics

revealed by mantel test.

Factors Statistic (r) Sig. (P) Statistic (r) Sig. (P)

River A River B

pH -0.27 0.89 -0.067 0.59

DO 0.67 0.006** -0.067 0.59

ORP 0.82 7.00E-04** -0.026 0.46

COD 0.45 0.02* 0.15 0.18

BOD 0.38 0.02* 0.22 0.10

NH4+-N -0.04 0.43 -0.12 0.73

TP 0.43 0.04* -0.13 0.75

WC 0.63 0.003** -0.057 0.56

Note: Boldface values indicate significant correlation (*P < 0.05; **P < 0.01).

34
Highlights:

 MBSR is used for the remediation of two black and stinking urban rivers.

 Water quality of the rivers is significantly improved after MBSR treatment.

 Aerobic microbes responsible for pollutant removal are stimulated.

 The ecological mechanism of MBSR restoration is elucidated.

35
Graphical abstract

36