Journal of Hazardous Materials 365 (2019) 483–493

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Journal of Hazardous Materials

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The collaborative effect of Chlorella vulgaris-Bacillus licheniformis consortia T

on the treatment of municipal water
Xiyan Jia, Huimin Lia, Jibiao Zhanga, , Hexige Saiyinb, Zheng Zhenga,
⁎ ⁎

a
Department of Environmental Science and Engineering, Fudan University, Shanghai, 200433, PR China
b
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, 200433, PR China

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Keywords: In this study, the effects of nutrient and dissolved organic matter removal, stress resistance (DNA methylation),
Algal-bacterial consortia and the algae-bacteria dynamic ratio of algal-bacterial consortia in actual municipal wastewater were in-
Municipal wastewater vestigated. Results indicate that the presence of a Chlorella vulgaris-Bacillus licheniformis consortium had pro-
Nutrients removal found effects. The removal rates of total nitrogen, ammonium, orthophosphate phosphorus and chemical oxygen
DNA methylation
demand were 88.82%, 84.98%, 84.87% and 82.25%, respectively. Protein-like substances, which are difficult to
degrade in the natural water environment, were significantly degraded in actual municipal wastewater.
Furthermore, the microbial diversity was measured. The algal-bacterial consortium did not disrupt the microbial
in-situ diversity of the actual municipal wastewater under suitable conditions. The global nuclear DNA me-
thylation level peaked at 7.80%. These results help to understand the effects of algal-bacterial consortia on
nutrient and pollutant removal and adaptability in actual municipal wastewater.

1. Introduction
Municipal wastewater pollution is becoming increasingly proble-
matic in large cities and could have a major negative impact on usable
water for urban residents. In addition to containing suspended solid
(SS) and nutrients, municipal wastewater contains a wide range of
other pollutants [1–3] that will cause harm and damage to the health of
residents and the waterbody. Black odorous water is one of the mani-
festations of extremely polluted municipal wastewater [4]. Alarmingly,
most cities in China have serious black odorous water problems [4].
The Qiujiang River is the most representative of a polluted municipal
river in Shanghai, with the typical features of black and odorous water

⁎
Corresponding authors.
E-mail addresses: jbzhang@fudan.edu.cn (J. Zhang), zzhenghj@fudan.edu.cn (Z. Zheng).

https://doi.org/10.1016/j.jhazmat.2018.11.039
Received 7 April 2018; Received in revised form 28 October 2018; Accepted 10 November 2018
Available online 13 November 2018
0304-3894/ © 2018 Elsevier B.V. All rights reserved.
X. Ji et al.
[5]. Therefore, the severe situation of this polluted municipal river
forced us to develop techniques to improve the water quality and
ecological regime. One of the most important tasks was to effectively
reduce the concentration of nutrients and chemical oxygen demand
(COD). Simultaneously, pollutants that are difficult to degrade in nat-
ural water should be reduced during the nutrient removal process. New
biological methods were selected to satisfy these two purposes. And the
organism which was added into the municipal wastewater should
normally survive. Furthermore, the new biological methods should not
disrupt the in-situ ecological structure and diversity.
As an algal genus that has a fast growth rate and short generation
time, Chlorella exhibits great potential in wastewater treatment [6].
Additionally, the genus of Chlorella can tolerate the rigorous environ-
mental conditions and efficiently assimilate nitrogen and phosphorus
from wastewater [7]. It is well known that a multitude of traditional
wastewater treatments have been used to improve the water quality of
the polluted municipal river. However, most of these technologies were
complicated and costly. Using algae to treat polluted municipal was-
tewater is economical. For the past few years, many researchers have
studied on treating municipal wastewater suing algae. High rate algal
pond (HRAP) reactors which adding carbon dioxide (CO2) are simple
reactor systems. The removal of nutrients and energy from wastewater
containing high levels of biomass were obtained [8]. On this basis, a
combined treatment using algae and bacteria can be regarded as an
advancement of the HRAP. Therefore, algal-bacterial consortia sym-
biotic systems has attracted significant research interest and many
studies have been carried out on this topic [9]. Research by Dr. De-
Bashan’s group found that the growth of Chlorella sorokiniana (C. sor-
okiniana) was promoted by Azospirillum brasilense (A. brasilense). The
removal rates of ammonium (NH3-N) and Phosphorus (P) by this algal-
bacterial consortium were 100% and 75%, respectively [10]. Further-
more, the ideal carbon sources for growth of heterotrophic Chlorella
vulgaris in sterilized municipal wastewater are Na-acetate and D-glucose
[11]. It has also been reported that during their interaction, C. sor-
okiniana exuded enough tryptophan and thiamine to promote the pro-
duction of indole-3-acetic acid (IAA) by A. brasilense [12]. Recent re-
search indicates that C. sorokiniana could be remotely induced by the
PGPB A. brasilense and Bacillus pumilus, thereby proposing a new
paradigm on how PGPB promotes microalgal growth, which might
serve to improve the performance of the Chlorella genus in biotechno-
logical applications [13]. Studies have also been performed on the
mechanistic model for reclamation of industrial wastewaters using
algal-bacterial photobioreactors, providing a useful tool to understand
the complex relationships between algae, bacteria, light and pollutant
concentrations [14]. However, the studies above just touched on the
characteristics and effects of nutrient removal by algae-bacteria con-
sortia in synthetic wastewater. As for the treatment of actual municipal
wastewater, the research focused on the removal effect and mechanism
of certain harmful substances in actual municipal wastewater. For ex-
ample, some researchers studied the removal of fragrances and orga-
nophosphate flame retardants in municipal wastewater treatment plant
effluents [15]. A method combining nanofiltration and ozonation for
the remediation of real municipal wastewater effluents has also been
studied [16]. Additionally, the kinetics and microbial ecology of batch
sulfidogenic bioreactors for co-treatment of actual municipal waste-
water were studied in previous research [17]. Other studies also in-
vestigated the fate of amisulpride, sulpiride and lamotrigine in muni-
cipal wastewater treatment plants undergoing biological treatment
[18]. However, it is rare to study the use of algal-bacterial consortia
systems to reduce the concentration of nutrients and other dissolved
organic matter (DOM) in actual municipal wastewater. Furthermore,
few researchers have studied and evaluated the adaptability and stress
resistance of algal-bacterial symbionts in actual municipal wastewater,
and fewer still have quantified this adaptability and stress resistance
from the perspective of DNA methylation. The symbiotic adaptability of
algae and bacteria in municipal wastewater systems still limits their
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Journal of Hazardous Materials 365 (2019) 483–493
applications in actual wastewater [19].
In the present study, a new method was proposed to study the
morphological features and characteristics of nutrients and DOM re-
moval. In addition, the effects of an algal-bacterial consortium on in-
situ microbial diversity, dynamic bacterial-algal gene copy ratios,
adaptation and resistance of the consortium in actual municipal was-
tewater were investigated. This study aimed to explore the performance
of a Chlorella vulgaris-Bacillus licheniformis symbiotic system for re-
moving pollutants from municipal wastewater under different condi-
tions. Laboratory scale analysis of total nitrogen (TN), NH3-N, ortho-
phosphate phosphorus (PO43−-P), COD and DOM was performed by
analyzing three-dimensional excitation and emission matrices
(3DEEM), microbial diversity, dynamic bacterial-algal gene copy
numbers and global nuclear DNA methylation (5-mC) levels. These
results can help to facilitate the use of algal-bacterial consortium for
improving the water quality of municipal wastewater.
2. Materials and methods
2.1. Water samples and microorganisms
Water samples were collected from seven sites distributed
throughout the Qiujiang River, a typically polluted municipal river in
the Yangpu District of Shanghai, China (Fig. S1). A 5-L vertical plex-
iglass water sampler was used to collect fresh wastewater, which was
then transferred to a sterile glass sample bottle and placed into a
Styrofoam box containing ice packs. All water samples were uniformly
mixed. The water quality indices of the fresh municipal wastewater are
shown in the supplementary materials (Table S1). Solids in the water
samples reduce the light intensity, which directly impacts the photo-
synthetic process. Therefore, all water samples used in this study were
filtered through 0.22 μm polyether sulfone (PES) filters prior to segre-
gating the solids.
Chlorella vulgaris (No. FACHB-8) (C. vulgaris) was obtained from the
Freshwater Culture Collection at the Institute of Hydrobiology (Wuhan,
China). Bacillus licheniformis (No. 1.7461) (B. licheniformis) was ob-
tained from the Institute of Microbiology, Chinese Academy of Sciences
(Beijing, China). The algae inoculums were cultivated in 1000-mL
conical flasks containing 400 mL BG-11 medium under a light intensity
of 120 μmol/(m2 s) at 28 ± 1℃ for 7 days. The cell concentration of C.
vulgaris was quantified by the hemocytometer method using an optical
microscope. The relationship between the algal cell concentration and
optical density of the algal solution at 680 nm (OD680) was established.
This allowed for calculation of algal cell concentrations by measuring
the OD680 of the algae solution according to the linear relationship. The
bacterial cells were cultured in a 1000-mL conical flask containing
400 mL of LB medium at 28 ± 1℃ for 7 days with a 12 h/12 h light/
dark cycle at a light intensity of 120 μmol (m2 s)−1. The algal-bacterial
consortium was cultured in a 500-mL conical flask containing 300 mL
fresh municipal wastewater with a 12 h/12 h light/dark cycle at a light
intensity of 120 μmol (m2 s)−1 and a rotary shaker speed of 120 rpm.
The temperature and pH conditions of control groups and experimental
groups are shown in Table 1. Descriptions of samples used in this study
are shown in Table 2. The initial concentrations of B. licheniformis and
C. vulgaris were kept at 1 × 105 cell mL−1 and 3 × 105 cell mL−1, re-
spectively.
Table 1
Descriptions of treatment conditions.
Conditions Temperature (℃) pH value With algal-bacterial consortia
C0 20 7.14 No
C1 4 5.12 Yes
C2 28 5.12 Yes
C3 4 7.14 Yes
C4 28 7.14 Yes
X. Ji et al. Journal of Hazardous Materials 365 (2019) 483–493

Table 2 10 nm for the excitation and emission. This method allows all the
Descriptions of municipal wastewater samples under different treatment con- fluorescence information within each region to be considered during
ditions. analysis [22].
Samples Temperature (℃) pH value With algal-bacterial Cultured days
consortia
2.5. Pretreatment for confocal laser scanning microscopy
T0 20 7.14 No 0
T1 20 7.14 No 12
T2 28 7.14 Yes 12 Samples of the algal-bacterial consortia were extracted from the
T3 28 5.12 Yes 12 reactors on the 12th day of the experimental period. The fresh samples
T4 4 7.14 Yes 12 were transferred to sterile 1-mL microcentrifuge tubes and then cen-
T5 4 5.12 Yes 12
trifuged at 8000 g for 60 s. The precipitate was washed three times with
phosphate buffer saline (PBS) solution. Thus, the bacteria and micro-
organisms that were suspended in the municipal wastewater were wa-
shed away. The precipitate was diluted with PBS using the vortex os-
cillator. The 50-μL mixed solution was placed on an adhesion
microscope slide (Brand, China, P/N 01-11215-50, 75 × 25 × 1.1 mm)
and allowed to dry. Next, 50 μL of 1% paraformaldehyde was dropped
onto the sample. Finally, the sample was observed by confocal laser
scanning microscopy (CLSM). The preprocessing operations were per-
formed at 22℃ under sterile conditions. And the preprocessing of
samples for differential-interference microscopy (DIC) were performed
using the same method.

2.6. DNA extraction and sequencing

Total DNA was extracted from the samples using the DNeasy Power
Soil Kit (QIAGEN, USA) according to the manufacturer’s instructions.
The extracted DNA was sent to Personalbio Co., Ltd. (Shanghai, China)
for 16S rRNA gene amplification, library construction and sequencing.

2.7. Quantification of dynamic bacteria-algae gene copies by q-PCR

Fig. 1. Schematic of the lab-scale algal-bacterial symbiosis system. Cells from the algal-bacterial consortia were harvested from 10-mL
samples by centrifugation at 8000 g for 15 min at 4 ℃. Total DNA was
immediately extracted from every sample using the DNeasy Power Soil
2.2. Experimental setup
Kit (QIAGEN, USA). The primers sets were specific for either algae or
bacteria. Primer sequences are shown in Table S2. The amplification
The experimental systems were placed in a sterile environment. The
efficiencies of the primers are shown in Table S3. The SYBR and mix-
system consisted of a reactor with a 500-mL conical flask, a shaking
ture A of primers is provided in Table S4. The composition of the
incubator (ShanZhi, China, TS-211GZ) and a sampling tap. The reactors
testing system for qPCR assays is shown in Table S5. The cycling
for the experimental groups contained C. vulgaris, B. licheniformis (with
conditions included an amplification step at 95 ℃ for 15 min to activate
the defined ratio of algae and bacteria) and 300 mL of fresh municipal
the DNA polymerase, followed by 40 cycles of denaturation at 95 ℃ for
wastewater (Fig. 1).
15 s and annealing at 60 ℃ for 30 s.

2.3. Water quality detection

A 15-mL algal-bacterial solution was centrifuged at 3600 ⋅ g for
10 min at 25℃, after which the supernatant was filtered through a
0.22 μm PES membrane. The filtrate was used to determine physical-
chemical parameters such as TN, NH3-N, PO43−-P and COD. These
nutrient concentrations were measured according to the methods out-
lined by American Public Health Association [20].
2.8. Global nuclear DNA methylation (5-mC) levels of microorganism in the
algae-bacteria symbiotic system
Total DNA of cells were isolated from the algae-bacteria symbiotic
system. This was then used to determine the global methylation level
using the MethylFlash™ Global DNA Methylation (5-mC) ELISA Easy Kit
(Colorimetric, USA) [23], according to the manufacturer’s instructions.

2.4. Fluorescence 3DEEM measurement

The wastewater samples were centrifuged at 1660 g and the su-
pernatant were passed through a 0.22 μm PES filter before testing.
Fluorescence spectra were obtained in triplicate using a spectrometer
(HORIBA Scientific Aqualog, Japan). The dilution ratio was determined
after measuring successive dilution ratios to limit overlapping signals.
The sample was diluted with Mili-Q ultrapure water to avoid inner filter
effects [21]. The scan ranges were set at 250–450 nm and 250–600 nm
for excitation and emission, respectively. The scan speed was fixed at
1000 nm·min−1 with a 2 nm increment, while the slit width was fixed at
2.9. Statistical analysis
All experiments were performed in triplicate and GraphPad Prism
software was used to process the data. Results are shown as the average
value ± standard deviation. Parametric one-way analysis of variance
(ANOVA) was used to examine significant differences among the groups
of samples. The Student’s t-test was used to determine significance
among the treatments. Points denoted by different lower-case letters
and capital letters on each curve differ significantly at P＜0.05 and P＜
0.01, respectively.

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X. Ji et al. Journal of Hazardous Materials 365 (2019) 483–493

Fig. 2. The CLSM images of algae-bacteria consortia cultured in municipal wastewater (A1, A2 and A3 were for sample the Chlorella vulgaris cultured individually in
municipal wastewater; B1, B2 and B3 were for sample T2; C1, C2 and C3 were for sample T4; D1, D2 and D3 were for sample T3; E1, E2 and E3 were for sample T5).

3. Results and discussion
3.1. CLSM images of algal-bacterial consortia cultured in municipal
wastewater
The CLSM and DIC images of algal-bacterial consortia cultured in
municipal wastewater under different treatment conditions are shown
in Fig. 2.
The algal-bacterial consortium was cultured in municipal waste-
water at condition of 28℃ and pH 7.14. The adherence of bacteria on
the surface of the algae was abundant. Therefore, the cytoderm outline
was indistinct (Fig. 2B1-3). However, when the algal-bacterial con-
sortium was exposed to a stress environment, such as low temperature
and low pH, the shape and features of the consortium changed. The
standard features of microalgal-bacterial aggregates under normal
temperature and pH have been previously demonstrated using SEM [9].
Images obtained in this study are consistent with the previously pub-
lished results, demonstrating ellipsoid microalgal-bacterial aggregates
(B1, B2 and B3 in Fig. 2). When the conditions changed to low tem-
perature and low pH, there was no obvious bacterial adherence on the
surface of the algae. Moreover, the shape of the algae became fusiform
(Fig. 2C1-3, D1-3 and E1-3), which increases survival in the stressed
environment and protects the cells from injury. Algal cells, such as C.
vulgaris and Microcystis aeruginosa, are able to stimulate some physio-
logical changes, including morphological changes, to resist stress from
unsuitable growth environments (such as ozone stress) [24,25]. It is

486
X. Ji et al.
reported that the cell walls of some algae that were exposed to chemical
or physical radiation and other stress conditions can change their
structural components and surface shape of the cell walls, which can be
regard as protective mechanism against adverse conditions in the ex-
ternal environment [26]. Additionally, A1, A2 and A3 were for sample
the C. vulgaris cultured individually in municipal wastewater. Results
show that the algal-bacterial consortium could survive in natural mu-
nicipal wastewater at 28℃ and pH 7.14; however, a reduction in tem-
perature and pH had a negative effect on the growth of the algal-bac-
terial consortium.
3.2. Water quality analysis
Water quality indices were applied as an established method to
assess the function of the algal-bacterial consortia symbiotic system in
municipal wastewater management [27,28]. These indices were used to
evaluate the removal efficiencies of TN, NH3-N, PO43−-P and COD by
the algal-bacterial consortium from municipal wastewater. The removal
rates of TN, NH3-N, PO43−-P and COD were 88.82%, 84.98%, 84.87%
and 82.25%, respectively, at 28℃ and pH 7.14, which are en-
vironmentally relevant conditions for effective growth of the algal-
bacterial consortium.
The established trends in nutrient removal are demonstrated in
Fig. 3, showing that all samples exhibited a significant decrease in the
concentrations of TN, NH3-N, PO43−-P and COD. In accordance with
previous studies, the removal rates of ammonium, nitrate, nitrite were
75%, 6% and 36%, respectively [29]. The COD removal efficiency in a
previously published study that used an algal biofilm to treat domestic
wastewater was 80.2% [27]. In this study, the COD removal efficiency
was 82.5%. Furthermore, nutrient removal in the previous study was
also slightly lower than that of this study. This can be attributed to the
fact that the presence of bacteria enhanced the biological activity of the
algae. Simultaneously, a portion of the COD, as well as some of the
nitrogen and phosphorus nutrients in the municipal wastewater, were
consumed by the biological activity of the bacteria [30,31]. Compared
with single algae, algal-bacterial consortia play an important role in
reducing COD and nutrients in the wastewater. Moreover, the lowest
concentrations of TN, NH3-N, PO43−-P and COD were observed on the
6th day during the log phase of C. vulgaris growth. This is because the
logarithmic phase is the most vigorous period of algal activity [32].
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Journal of Hazardous Materials 365 (2019) 483–493
These findings show that the algal-bacterial consortium could fulfill the
requirements for effective elimination of nutrients from municipal
wastewater.
3.3. Characteristics and removal of DOM
DOM is a heterogeneous mixture of aromatic and aliphatic organic
compounds containing oxygen, nitrogen and sulfur functional groups
[33]. During wastewater treatment, DOM affects biogeochemical pro-
cesses, particle stability, particle transport and metal complexation,
among other aspects [34]. In this study, a synchronous absorption 3D
fluorescence spectrometer, coupled with origin fluorescence spectro-
metry, was used to automatically trace quinine sulfate to correct the
internal filtering effects and the Rayleigh and Raman scattering lines
[35]. Matrix data were directly analyzed using Parallel Factor Analysis
(PARAFAC). PARAFAC could be used to decompose the complex com-
ponents of DOM mixtures. Stochastic mathematical processes was used
to define the different components and sources of DOM [22]. Integra-
tion was carried out over the specific spectral region that corresponded
to the specific fluorophores bound to fluorescent compounds. The
3DEEM data in this study was divided into four areas according to the
results of the PARAFAC. The fluorophore in region IV was a protein-like
substance, which is in accordance with previously reported findings
[35].
The humification index (HIX) is used to characterize the degree of
DOM humification. HIX is defined as the area of the emission peak in
435–480 nm divided by the area of the peak in 300–345 nm [36]. The
high value of HIX means the degree of DOM humification was high.
These indicated that the stability and environmental persistence were
high. In general, HIX values from land-sourced DOM are higher than
those from water bodies or water treatment blowdown [37]. The HIX is
calculated as shown in Eq. (1). The biological index (BIX) is used to
indicate the contribution of each DOM source to in-situ biological ac-
tivity [38]. It is defined as the fluorescence intensity at the emission
wavelength 380 nm, divided by the fluorescence intensity at the emis-
sion wavelength of 430 nm, at an excitation wavelength of 310 nm
[38]. High values of BIX (values greater than 1) [39] obtained using this
calculation suggest that DOM is mostly from an endogenous microbial
origin within the water body. If the BIX value is low (from 0.6 to 0.7)
[39], the DOM source is mainly from terrestrial and exogenous release
Fig. 3. Changes of TN, NH3-N, PO43−-P and
COD concentration in municipal wastewater
under different treatment conditions. Points
denoted by different lower case letters and
capital letters on each curve differ significantly
at P＜0.05 and at P＜0.01 with ANOVA, ac-
cording to Student’s t-test. Bars represent
standard error. C0 represents the treatment
condition of 20℃ and pH 7.14, without the
algal-bacterial consortia (Control group). C1
represents the treatment condition of 4℃ and
pH 5.12, with the algal-bacterial consortia
(Experimental group). C2 represents the treat-
ment condition of 28℃ and pH 5.12, with the
algal-bacterial consortia (Experimental group).
C3 represents the treatment condition of 4℃
and pH 7.14, with the algal-bacterial consortia
(Experimental group). C4 represents the treat-
ment condition of 28℃ and pH 7.14, with the
algal-bacterial consortia (Experimental group).
X. Ji et al.
[40]. The BIX is calculated as shown in Eq. (2).
f435
HIX = 480
( ex = 255nm)
f300 345 + f435 480 (1)
f380
BIX = ( ex = 310nm)
fmax 420 (2)
435
Based on the analytical data calculated by Eq. (1) and Eq. (2), the
range of HIX and BIX in this study was 0.21-0.34 and 0.52-0.76, re-
spectively. The excitation wavelengthλex in Eq. (1) was 225 nm. This
indicates that the original source of DOM in the municipal wastewater
was from wastewater discharge rather than in-situ microorganisms. The
source of DOM is different and its degradation effect will be different
[41]. Estimating the source of DOM in municipal wastewater helps to
accurately identify the source of organic pollutants. Thus, we can re-
duce pollution from the source. The experimental results in this study
indicate that the source of DOM is mainly exogenous pollutant emis-
sions rather than aquatic endogenous organisms. This can be used to
reduce DOM pollution by reducing the source of emissions when
managing municipal wastewater [42].
The PARAFAC analysis not only identifies the main constituents of
DOM in municipal wastewater but also their relative quantitative in-
tensity (Fig. 4). In this study, the fluorescence of the quinine sulfate
equivalent (QSE) was used to measure the relative intensity of protein-
like substances in the municipal wastewater. The results demonstrate a
relative protein-like removal rate up to 56.4% in sample T2, which was
significantly higher than that of samples T1, T3, T4 and T5.
Results suggest that the presence of an algal-bacterial consortium
had a profound effect on the removal efficiency of protein-like sub-
stances. The initial concentration of protein-like substance was high
(Fig. 4, Sample T0). By the end of the operational period, the con-
centration of protein-like substance remained high (Fig. 4, Sample T1),
suggesting that they were difficult to degrade in the natural environ-
ment. However, when the conditions were changed to low temperature
and pH, growth of the algal-bacterial consortium was inhibited. The
removal efficiency of protein-like substance was reduced (Fig. 4,
Sample T3 and T5). Therefore, the protein-like substances in municipal
wastewater would not be effectively eliminated by an algal-bacterial
consortium under stressful environmental conditions. These results in-
dicate that a high-stress environment, such as low pH and low tem-
perature, would have an adverse effect on the survival of algal-bacterial
consortia, subsequently reduced the removal of protein-like substances.
The above results indicate that once the environmental conditions are
appropriate, algal-bacterial consortia can effectively remove protein-
like substances that are difficult to degrade in the natural environment.
3.4. Influence of conditions on the diversity of the microbial community
Illumina MiSeq high-throughput sequencing of the 16S rRNA gene
was used to investigate the influence of the in-situ microbial commu-
nity composition and the variation in mechanisms of the algal-bacterial
consortium cultured in municipal wastewater under different condi-
tions. The V3-V4 hypervariable region of the 16S rRNA gene was se-
quenced for DNA extracted from samples [43]. These sequences were
classified into a total of 6500 OTUs at a 97% similarity threshold [43].
In the horizontal direction, with increasing OTU number, the width of
the curve became larger (Fig. 5a), suggesting that the richness of mi-
crobial abundance was high [44].
Flatter curves showed a higher degree of evenness in microbial
composition (Fig. 5a) [45,46]. The trend observed for the untreated
water without the algal-bacterial consortium was the lowest of all the
five curves. This indicates that the degree of evenness of the microbial
community composition was the highest among the five sample types.
Simultaneously, the horizontal width of the untreated water curve was
also the longest, revealing that the microbial diversity of the untreated
water was high. However, water quality indices, such as TN, NH3-N and
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Journal of Hazardous Materials 365 (2019) 483–493
PO43−-P, were critically high. When the algal-bacterial consortium was
present in municipal wastewaters (28℃, pH 7.14), the water quality
improved compared to that of the untreated water at the end of the
experimental period. These results suggest that the presence of the
algal-bacterial consortium did not disturb the in-situ microbial diversity
of the municipal wastewater under suitable conditions. Meanwhile, not
disrupting the in-situ microbial diversity can be regarded as one of the
manifestations of the algal-bacterial consortium’s adaptation to muni-
cipal wastewater [47]. This also indicates that suitable temperature and
pH are beneficial for the algal-bacterial consortium to remove nutrients
and improve water quality.
Based on the 241,697 sequences randomly selected from each of the
four samples, there were significant differences observed in alpha (α)-
diversity indices; observed species; and Simpson, Shannon [48] and
Chao1 [49] indices (Table 3). The Chao index was high in samples T1
and T2, indicating that more bacteria proliferated in these two systems.
Furthermore, the Shannon index of sample T2 (6.92) was higher than
that of samples T3 (5.95), T4 (6.09) and T5 (4.74), demonstrating that a
stressful environment with low temperature and pH decreased species
diversity in the microbial community.
These results suggest that municipal wastewater without the algal-
bacterial consortium had the highest microbial diversity of all 5 sam-
ples. Differences in the microbial diversity of these samples were sig-
nificant, with the distribution and dominant bacteria showing distinct
variation [50,51].
At the phylum-level, Proteobacteria dominated the bacterial com-
munity in 4 of the samples, while Firmicutes dominated sample T4
(Fig. 5b). A high number of Cyanobacteria (30%) and Actinomycetes
(23.7%) proliferated in sample T1. Cyanobacterial growth can result in
biomass blooms [52], while (Jin et al. 2017)Actinomycetes, another
phylum of harmful bacteria, are widespread in municipal wastewater
[53,54]. Compared to the algal-bacterial consortia system observed in
sample T2, the wastewater samples without the algal-bacterial con-
sortium contained more Cyanobacteria and Actinomycetes. This indicates
that the C. vulgaris-B. licheniformis consortium could inhibit Cyano-
bacteria and Actinomycetes in polluted municipal wastewater and that
the algal-bacterial consortium could reduce the risk of Cyanobacterial
blooms. It has been established that the growth of M. aeruginosa, be-
longing to the phylum Cyanobacteria, could be inhibited by a glycolipid-
type compounds from Bacillus subtilis [55]. For sample T3, the abun-
dance of Cyanobacteria increased significantly compared to that of
sample T2, while Firmicutes dominated (80%) in sample T4, with the
microbial diversity of this system significantly reduced. In sample T5, it
was observed that Proteobacteria (95%) was the exclusive phylum, thus,
at this stage microbial diversity was lost. These results show that low
pH environments have a negative effect on the growth of algal-bacterial
consortia. Furthermore, pH conditions have a more significant effect
than temperature on the growth of algal-bacterial consortia and their
ability to remove nitrogen and phosphorus nutrients and DOM. In cases
where the microbial diversity was disrupted and single species phe-
nomenon occurred, all water quality indices were at the same level as
that of the untreated municipal wastewater.
In conclusion, the algal-bacterial consortium could survive in mu-
nicipal wastewater and simultaneously eliminate N and P nutrients and
DOM at 28 ℃ and pH 7.14. Furthermore, the fatal stress factor for the
algal-bacterial consortium and microbial diversity of the system was
pH, rather than temperature. Normal microbial species diversity was
maintained when the algal-bacterial symbiotic consortium was cultured
at 28 ℃ and pH 7.14. In comparison, the stressed environment of
sample T5 suppressed the normal growth of the algal-bacterial con-
sortium, with simultaneous loss of microbial diversity under environ-
mental stress conditions.
Clustering analysis was performed on sample data at the genus-level
and the results are demonstrated with heat maps [56]. High and low-
abundance populations could be distinguished, allowing interpretation
using a color gradient to demonstrate the similarity of community
X. Ji et al.
Fig. 4. Fluorescence excitation-emission matrix spectra of the wastewater samples under different treatment conditions (All the samples were diluted 10 times with
the Mili-Q ultrapure water).
distribution in different samples [57]. In Fig. 5c, red represents the
genera with high abundance in the corresponding sample, while green
represents the genera with low abundance. For sample T1, the genera
with high abundance were Achromonas, Faecalibacter, Fusucatenbacter,
Escherichia-shigella, Akkermansia, Lachnospiraceae, Blautia, Bacteroide,
Subdoligranulum, Bifidobacterium, Ruminococcus-2, Sediminbacterium,
Methylobacterium, Amycolatopsis, Ochrobacterium, and Vibrionimonas.
The genera with the highest abundances were distinctively different
and the presence of the algal-bacterial consortium significantly changed
the microbial community structure of the polluted municipal river
wastewater.
3.5. The dynamic gene copy ratios of bacteria and algae
The quantification of specific genetic markers by qPCR is considered
a precise and sensitive method [58,59]. Despite of the diminishment of
dead cells during the process of centrifugal collection and the variations
of plastid genome copies in different environments [60,61], the DNA of
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Journal of Hazardous Materials 365 (2019) 483–493
the microorganism was extracted for the qPCR assay.
The qPCR results suggest that the dynamic gene copy ratios of mi-
crobes in the algal-bacterial consortia symbiotic system were sig-
nificantly different under the different treatment conditions. In sample
T2, the bacterial-algal gene copy ratio increased gradually during the
first 6 days, with the peak level observed on the 6th day, which is
potentially during the log phase of the C. vulgaris. This is attributed to
the fact that algae are extremely active in various activities during the
logarithmic growth phase, as mentioned earlier [32]. The bacterial-
algal gene copy ratios increased to a maximum of 3987:1. In a previous
study, the relative algal-bacterial ratio peak was on the 4th day [60],
while after the 6th day the bacterial-algal gene copy ratio decreased
until the end of the operational period. Furthermore, the T2 bacterial-
algal gene copy ratios were distinctly lower than that of sample T4. In
samples T3 and T5, the bacterial-algal gene copy number ratio was very
low, with no fluctuations, as was also observed in the untreated water
without the algal-bacterial consortium (Fig. 6a). The presence of the
algal-bacterial consortium appeared to be supported by some other
X. Ji et al. Journal of Hazardous Materials 365 (2019) 483–493

Fig. 5. a) The abundance rank of wastewater samples under different treatment conditions. b) Phylum-level taxonomic distribution of wastewater samples under
different treatment conditions. c) Hierarchical clustering analysis of bacterial communities in wastewater samples under different treatment conditions at genus level.

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X. Ji et al. Journal of Hazardous Materials 365 (2019) 483–493

Table 3 would have profound effects on the performance of nutrient removal

Summary of 16S rRNA Illumina MiSeq sequences, operational taxonomic units systems, which is in accordance with results of the published study
and microbial α diversity indices of the samples. [60].
Symbols Sequences Operational Species Simpson Shannon Chao1
taxonomic units 3.6. Global nuclear DNA methylation of the alga-bacteria symbiotic system

T1 40271 6600 61 0.96 7.83 1640

Dynamic DNA methylation patterns are frequently employed to il-
T2 85820 5300 57 0.97 6.92 1229
T3 39568 4800 49 0.90 5.95 1007 luminate the environmental stress of organisms [63]. Environmental
T4 31842 3700 42 0.94 6.09 784 signals can trigger variations in genome-wide DNA methylation pat-
T5 44196 2600 34 0.86 4.74 654 terns, which influences cell responses to stress conditions through gene
regulation [64]. The stress tolerance of the algae-bacteria symbiotic
system is related to changes in DNA methylation [65]. The influence of
varying conditions on DNA methylation levels of microorganisms in the
algae-bacteria symbiotic system were investigated in the present study.
The DNA methylation (5-mC) value of the untreated wastewater system
(Sample T1) was 2.92% (Fig. 6b). The 5-mC level of the system har-
boring the algal-bacterial consortium (Sample T2) under appropriate
environmental condition was 7.80%. The 5-mC level of the system that
harbored the algal-bacterial consortium (Sample T3) under stressed
conditions (low temperature and pH) was 3.71%.
Comparing the results of these three groups, it can be found that the
system without the algal-bacterial consortium has the weakest ability to
adapt to a stressed environment. The system with the algal-bacterial
consortium showed the strongest stress resistance to adapt stressed
environment under suitable conditions. The stress resistance of the
algal-bacterial consortium is significantly reduced when the environ-
ment becomes highly rigorous. Nonetheless, the stress resistance of the
system within the algal-bacterial consortium is still better than that of
the system without the addition of the algal-bacterial consortium.
Stressed environments are an adverse condition that harmfully af-
fect the development of organisms. After exposure to stress, such as low
temperature and pH, organisms try to adapt to changing conditions by
altering metabolic rates, signal pathways and the expression of adaptive
genes [65].

4. Conclusions

In this study, the presence of a C. vulgaris-B. licheniformis consortium

Fig. 6. a) The dynamic bacteria-algae gene copy ratios of wastewater samples
under different treatment conditions. Points denoted by different lower case
letters and capital letters on each curve differ significantly at P＜0.05 and at P
＜0.01 with ANOVA, according to Student’s t-test. Bars represent standard
error. C0 represents the treatment condition of 20℃ and pH 7.14, without the
algal-bacterial consortia (Control group). C1 represents the treatment condition
of 4℃ and pH 5.12, with the algal-bacterial consortia (Experimental group). C2
represents the treatment condition of 28℃ and pH 5.12, with the algal-bacterial
consortia (Experimental group). C3 represents the treatment condition of 4℃
and pH 7.14, with the algal-bacterial consortia (Experimental group). C4 re-
presents the treatment condition of 28℃ and pH 7.14, with the algal-bacterial
consortia (Experimental group). b) DNA methylation (5-mC) levels of total
microorganism in the algae-bacteria symbiotic system samples under different
treatment conditions. Columns denoted by a different lower case letter differ
significantly by ANOVA at P＜0.05. Pairs of columns denoted by asterisks differ
significantly with Student’s t-test at P＜0.01. Bars represent standard error.
microbe in the municipal river, especially for sample T2. Correspond-
ingly, the water quality index of sample T2 was better than that of the
other samples. The issue of algal-bacterial population dynamics in
HRAPs has been previously studied. The function of bacteria in algal-
bacterial symbiotic systems is to supply the carbon dioxide (CO2) and
organics for algal growth [62]. Therefore, the bacterial-algal ratio
has positive effects on nutrients removal and DOM pollutant reduction
in municipal wastewater. This treatment processes reduced the con-
centration of TN, NH3-N, PO43−-P and COD in the wastewater at high
removal rates. Protein-like substances that are more difficult to degrade
in nature are effectively degraded in algal-bacterial symbiosis systems.
The algal-bacterial consortia can survive normally in natural municipal
wastewater and have stress resistance to stressed environments with
low pH value and low temperature; however, single-algae and single-
bacteria systems without this stress resistance. The presence of the
algal-bacterial consortium not only preserves the in-situ microbial di-
versity of the natural municipal wastewater, but it is also conducive to
maintaining the stability of the microbial community structure in mu-
nicipal wastewater. This will aid the application of algal-bacterial
symbiotic systems for remediation of polluted municipal rivers.
Acknowledgments
The study was supported by Guizhou Science and Technology
Department, China (No. Qiankehezhicheng (2017) 2859).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jhazmat.2018.11.039.
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