Impact of Effective Microorganisms and Chlorella Vulgaris On

sustainability
Article
Impact of Effective Microorganisms and Chlorella vulgaris on
Eriocheir sinensis and Water Microbiota in Ponds Experiencing
Cyanobacterial Blooms
Jiancao Gao 1,2,3,† , Nailin Shao 2,† , Yi Sun 2 , Zhijuan Nie 2 , Xiwei Yang 3 , Fei Dai 3 , Gangchun Xu 1,2, *,‡
and Pao Xu 1,2, *,‡
1 Wuxi Fisheries College, Nanjing Agricultural University, Wuxi 214081, China; gaojiancao@ffrc.cn
2 Key Laboratory of Integrated Rice-Fish Farming Ecology, Ministry of Agriculture and Rural Affairs,
Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China
3 Jiangsu Noah’s Ark Agricultural Technology Co., Ltd., Changzhou 213147, China
* Correspondence: xugc@ffrc.cn (G.X.); xup@ffrc.cn (P.X.); Tel.: +86-0510-85390029 (G.X.)
† These authors contributed equally to this work.
‡ These authors contributed equally to this work.
Abstract: Cyanobacterial blooms threaten the quality and safety of the Chinese mitten crab
Eriocheir sinensis. A combination of microalgae and probiotics seems a promising way to prevent and
control cyanobacterial blooms in aquaculture ponds. In E. sinensis cultivation, however, a related
strategy is still lacking. To assess the potential combined effects of effective microorganisms (EM) and
Chlorella vulgaris on regulating cyanobacterial blooms, in this study, we detected the alterations in the
physiology of E. sinensis, as well as water quality and microbial compositions of E. sinensis culture
ponds with cyanobacterial blooms. As a result, supplementary EM and C. vulgaris had no adverse
effects on the growth or digestive or antioxidant ability of E. sinensis but improved the water quality
of the pond by reducing total ammonia nitrogen and total nitrogen levels. We found an increase
in bacterial diversity and evenness, while a decrease in the diversity of fungal and phytoplankton
communities was related to supplementary EM and C. vulgaris. Interestingly, EM coupling C. vulgaris
promoted the restoration of the bacterial and fungal community composition in cyanobacterial
Citation: Gao, J.; Shao, N.; Sun, Y.;
blooms ponds, particularly the increase of Mychonastes abundance and the decrease of Cyclotella. This
Nie, Z.; Yang, X.; Dai, F.; Xu, G.; Xu, P.
Impact of Effective Microorganisms
study laid the foundation for the prevention and control of potential risks in aquaculture.
and Chlorella vulgaris on Eriocheir
sinensis and Water Microbiota in Keywords: microbial community; cyanobacterial blooms; risk prevention and control; Eriocheir sinensis
Ponds Experiencing Cyanobacterial
Blooms. Sustainability 2023, 15, 7362.
https://doi.org/10.3390/su15097362
1. Introduction
Academic Editors: Domitilla Pulcini,
Giulia Secci and Fabrizio Capoccioni The Chinese mitten crab, Eriocheir sinensis (H. Milne Edwards, 1853), is an endemic
economic aquaculture species in China [1]. Owing to the great demand among a growing
Received: 23 March 2023 number of Chinese consumers, it is widely cultured in China except for a few regions such
Revised: 21 April 2023
as Tibet autonomous region and Hainan province. In 2021, the annual output of E. sinensis
Accepted: 26 April 2023
reached 808,274 tons [2]. Nowadays, the quality and safety of E. sinensis are the bottlenecks
Published: 28 April 2023
to hinder the green development of the E. sinensis industry. In the culture of E. sinensis,
cyanobacterial blooms are one of the important factors threatening the quality and safety
of E. sinensis [3,4].
Copyright: © 2023 by the authors.
In E. sinensis culture ponds, cyanobacterial blooms are commonly formed by the follow-
Licensee MDPI, Basel, Switzerland. ing genera, e.g., Microcystis, Cyanobium, Calothrix, Sphaerospermopsis and Cylindrospermopsis [1].
This article is an open access article It is well documented that cyanobacterial blooms and their secondary metabolites, cyanobac-
distributed under the terms and terial toxins (such as microcystins, anatoxin-a, retinoic acid, microviridins, anabaenopeptins,
conditions of the Creative Commons aeruginosins, microginins, piricyclamides, and cyanopeptolins) can cause a series of prob-
Attribution (CC BY) license (https:// lems in aquaculture, such as compromising water quality, altering microbial community
creativecommons.org/licenses/by/ assembly, toxic to aquatic animals, and even threatening to human health by bioaccumula-
4.0/). tion through food chains [5–12].
Sustainability 2023, 15, 7362. https://doi.org/10.3390/su15097362 https://www.mdpi.com/journal/sustainability

Sustainability 2023, 15, 7362 2 of 23
Cyanobacterial blooms often alter the bacterial community assembly in aquatic ecosys-
tems [12,13]. This is mainly due to the cyanobacterial-heterotrophic bacterial associa-
tions [14–16]. Specifically, cyanobacterial-heterotrophic bacterial associations play key
roles in providing key nutrients (carbon, nitrogen, phosphorous) and limiting micronu-
trients (iron and vitamin B12) to cyanobacteria [14,16], facilitating cyanobacterial species
succession in response to nutrient limitation [15].
In nature, algae live together with bacterial communities, and the interactions of algae-
bacteria balance the planktonic microbiome [17,18]. In general, algae-bacteria interactions
are facilitated by secreted compounds. Algae exude dissolved organic carbon that is
available to bacteria, and in return, the metabolites of bacteria support the growth of algae,
such as vitamins and/or recycled nutrients [19,20]. In addition to positive interactions,
negative interaction exists between algae and bacteria. e.g., the quorum-sensing of bacteria
impacted the cell division and mortality of algae [21].
To meet the needs of quality and safety of aquatic products under the premise of
environmental protection, in aquaculture, microalgae-bacteria balance provides a promising
way to prevent and control potential risks. Thus, probiotics and beneficial microalgae are
favored by farmers for their efficient and harmless [22,23]. However, the microalgae-
bacteria strategy for combating and preventing cyanobacterial blooms is still lacking.
In our previous studies, we found the beneficial effects of effective microorganisms
(EM) and Chlorella on E. sinensis culture, e.g., improving the water quality, as well as the
nutritional composition and flavor quality of E. sinensis [4]. Thus, in the present study, we
aimed to lay the foundation for the prevention and control strategies of cyanobacterial
blooms in E. sinensis cultivation. We analyzed the water quality and microbial composi-
tions of E. sinensis culture ponds with and without cyanobacterial blooms. Furtherly, we
investigated the regulation effect of EM and Chlorella vulgaris on the change of water quality
and microbial communities in cyanobacterial blooms ponds.
2. Materials and Methods

2.1. Experimental Design and Management
This experiment was conducted from 19 July to 26 July 2021 in the Yangzhong scientific
research base of the Freshwater Fisheries Research Center, Chinese Academy of Fishery
Sciences. A total of nine E. sinensis cultural ponds (23 m in length and 73 m in width)
were selected in this study. Three normal ponds without supplements were selected as
the control group (hereinafter NG), and 6 ponds with cyanobacterial blooms were evenly
divided into 2 groups, the supplements group of EM and Chlorella vulgaris (hereinafter CS)
and the non-supplement group (hereinafter CN). Hydrophytes, i.e., Elodea nuttallii and
Hydrilla varticillata, were planted in each pond, as reported in our previous study [4].
In the CS group, C. vulgaris was used every 2 days with a dose of 22.5 mL/m2 on the
1st, 3rd, and 5th days, respectively. Meanwhile, EM was used every 2 days with a dose of
3.0 mL/m2 on the 2nd, 4th, and 6th days, respectively. EM with a viable bacteria number
of 5.0 × 106 CFU/mL was purchased from Jiangsu Hengtai Environmental Protection
Technology Development Co., Ltd., Wuxi, China. C. vulgaris was prepared in our laboratory
with fresh sterile BG-11 medium, which was cultured in 4500 Lux light, 12-12 h light-dark
cycles, and 26 ◦ C constant temperature. High-density algal liquid with a logarithmic
growth stage was used for the experiment. The final used C. vulgaris was at a cell density
of 5.0 × 107 cells/mL.
During the experiment, crabs were in the intermolt period between the 3rd molting to
the 4th molting of the adult crab culture stage and fed with a formula diet at 17:00 once
a day. Hydrophytes were managed to ensure reasonable density and space. Dissolved
oxygen was controlled above 5.0 mg/L by a micropore oxygenation system. The depth
of water in the ponds was about 1.0 m. In the CS group, the cyanobacterial blooms were
significantly reduced, with an obvious decrease in emerald green film downwind of the
ponds on the 7th day of the experiment. Therefore, we set 7 days as the experimental period.
2.2. Sampling and Water Quality Detection

Sampling was conducted at the beginning (day 0) and end of the experiment (day 7).
Microbial samples were collected at 9:00 am, as described in the previous study [1]. Briefly,
a total of 20 L of surface water were obtained by water collecting from four corners
and mixing with an equivalent volume. Then, 0.5 L of water was filtered with 0.22-µm
polycarbonate membranes. Finally, the filter membranes were frozen in liquid nitrogen and
stored at −80 ◦ C until DNA extraction.
Simultaneously, the water temperature, pH, and dissolved oxygen (DO) of each pond
were measured using HACH HQ30D (Hach Company, Loveland, CO, USA). Water quality
parameters, i.e., total nitrogen (TN), total ammonia nitrogen (TAN), nitrate nitrogen (NO3 -
N), nitrite nitrogen (NO2 -N), and total phosphorus (TP), were detected as described in
the previous study [1]. The N/P ratio was calculated using total nitrogen divided by total
phosphorus.
After 24-h fasting, a total of 18 crabs (6 female crabs from each pond) were sampled
from each group every sampling time. After anesthetized on ice, crabs were first measured
the wet weights, carapace length and width; then, their hepatopancreas were collected
carefully. Those tissues were frozen in liquid nitrogen immediately and stored at −70 ◦ C
for further analysis.
2.3. Enzyme Activity and Antioxidant Parameters Assays

To reveal the potential physiological differences of E. sinensis among groups, we de-
tected digestive enzyme activity and antioxidant parameters in the hepatopancreas of
E. sinensis. Lipase (A054-2-1) and α-amylase (C016-1-1) in hepatopancreas were detected
as described by Dai et al. (2020). Superoxide dismutase (SOD; A001-3), catalase (CAT;
A007-1), glutathione peroxidase (GPX; A005) activity and malondialdehyde (MDA; A003-1)
were determined as described elsewhere [24]. All used kits were purchased from Nan-
jing Jiancheng Bioengineering Institute (Nanjing, China). Enzyme activity or antioxidant
parameters was calibrated with total protein (TP) concentration.
2.4. DNA Extraction, PCR Amplification, and Sequencing

Total microbial DNA was extracted using the HiPure Soil DNA Kits (Magen, Guangzhou,
China) according to the manufacturer’s protocols. The V3-V4 region of the 16S rDNA and the V4
region of the 18S rDNA were amplified by PCR as described in the previous study [1]. Primers
of 341F (50 -CCTACGGGNGGCWGCAG-30 ) and 806R (50 -GGACTACHVGGGTWTCTAAT-30 )
were used for 16S rDNA amplification, while 528F (50 -GCGGTAATTCCAGCTCCAA-30 )
and 706R (50 -AATCCRAGAATTTCACCTCT-30 ) were used for 18S rDNA amplification.
In the present study, 18 procaryotic sequencing libraries and 18 eukaryotic sequencing
libraries were constructed. Briefly, amplicons were extracted from 2% agarose gels, purified
using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA),
and quantified using ABI StepOnePlus Real-Time PCR System (Life Technologies, Foster
City, CA, USA) as described in our previous study [1]. Purified amplicons were pooled
in equimolar and paired-end sequenced (PE250) on an Illumina NovaSeq 6000 system
(Illumina Inc., San Diego, CA, USA). The raw reads were deposited into the NCBI Se-
quence Read Archive (SRA) database (BioSample accessions range from SAMN28097324 to
SAMN28097341 under the BioProject PRJNA835239).
2.5. Tags Assembly, Filtering, and Taxonomy Annotation

Bioinformatics analyses were described in our previous study [1]. Briefly, clean
reads were obtained by raw reads filtering [25]. The filtering criteria were no adapter
sequence, less than 10% of unknown nucleotides, or more than 50% of bases with quality
(Q-value) > 20. Then paired-end clean reads were assembled into raw tags [26]. Clean tags
were obtained after raw tag filtering under specific filtering conditions [27]. The filtering
conditions are as follows: (1) break raw tags from the first low-quality base site where
the number of bases in the continuous low-quality value (the default quality threshold
is ≤3) reaches the set length (the default length is 3 bp), (2) filter tags whose continuous
high-quality base length is less than 75% of the tag length. Then, clean tags of ≥97.0%
similarity were clustered into operational taxonomic units (OTUs) using UPARSE [28].
After chimera and singleton tags removal using the UCHIME algorithm [29], the highest
abundance effective tag was regarded as the representative sequence for each OTU cluster.
The representative OTU sequences were used for taxonomy annotation using the RDP
classifier [30] based on SILVA database version 132 [31].
2.6. Community Composition and Differentially Abundant Taxons

The relative abundance of species at each taxonomic level was calculated using the
number of annotated effective tags divided by the total number of effective tags in the sam-
ple. In the bacterial community, we specifically analyzed the composition of cyanobacterial
genera in each group. To show the changes in community composition in each group on the
initial and final days, the stacked bar plots were used to visualize the relative abundance of
microbes at a given taxonomical level.
The differentially abundant taxon (from phylum to species) between groups was
evaluated by linear discriminant analysis effect size (LefSe) [32]. The differentially abundant
taxon was considered significant with the absolute value of the linear discriminant analysis
(LDA) score >4.0. To clarify the microflora alteration associated with supplementary EM
and C. vulgaris, we analyzed the significance of differentially abundant taxon between the
CS0 and CS7 groups using Welch’s t-tests. To further explore the changes in ecological
function among groups based on microbial community profiles, we analyzed variations in
ecological functions and metabolic phenotypes of prokaryotes using FAPROTAX [33] and
Bugbase [34], respectively.
2.7. Alpha and Beta Diversity Analysis

The alpha diversity was assessed as described in our previous study [1]. OTU rar-
efaction curve and rank abundance curves were plotted in the R project ggplot2 package
(version 2.2.1). In beta diversity analysis, we conducted principal coordinates analysis
(PCoA) and ANOSIM test based on weighted unifrac distances.
2.8. Statistical Analysis

Normality and variance homogeneity of data were tested with Shapiro-Wilk and
Levene tests (α = 0.05), respectively. Statistical differences in water quality parameters
and alpha diversity indices were assessed using 1-way ANOVA and Duncan’s multi-
comparison (or Tamhane multi-comparison in case of the heterogeneity of variances)
for normally distributed data, while Kruskal–Wallis tests were used for non-normally
distributed data. For those significantly altered microbial taxa in CS groups, we furtherly
analyzed the correlation between microbial taxa using Spearman correlation analysis
based on the relative abundance in all samples. PcoA and ANOSIM tests of weighted
unifrac distances were conducted in the R project Vegan package (version 2.5.3). Spearman
correlation analysis was conducted in the R project psych package (version 2.3.3). Data are
expressed as means ± standard deviations (SDs) of the respective pond replications, and
p values < 0.05 were considered statistically significant.
3. Results
3.1. Water Quality Parameters
During the culture period, the water temperature ranged from 31.6 to 31.9 ◦ C. In all
detected E. sinensis rearing ponds, dissolved oxygen reached over 100% air saturation due
to photosynthesis activity. Different from other water quality parameters, NO3 -N and pH
had no significant differences among the groups.
In the NG and CN groups, levels of TAN and TN were consistent or increased during
the experiment period. In contrast, levels of TAN and TN in the CS7 group significantly
decreased than that in the CS0 group (Table 1). During the experiment period, NO2 -N had
a low level in all groups that ranged from 0.001 to 0.003 mg·L−1 (Table 1). Compared to
the initial levels, the 7-day NO2 -N level had a decreased trend in the NG and CS groups,
while that was increased in the CN groups (Table 1). Unlike the higher level of TP in the
NG7 and CN7 groups than their initial levels, the level of TP had no significant difference
between the CS0 and CS7 groups (Table 1). The NPR of the NG7 group was slightly lower,
albeit non-significant, than that of the NG0 group. Similarly, the NPR of the CS7 group was
significantly lower than that of the CS0 group. However, the NPR of the CN7 group was
significantly higher than that of the CN0 group (Table 1). Taken together, a supplement
of EM and C. vulgaris had an improved effect on the water quality of cyanobacterial
bloom ponds.
Table 1. Descriptive statistics of water quality parameters and their variations across different groups.
Values are presented as means ± SD. Statistically significant differences are indicated by different
letters (p < 0.05).
Water Quality Parameters NG0 NG7 CS0 CS7 CN0 CN7

Total ammonia nitrogen (TAN;
0.22 ± 0.02 c 0.20 ± 0.02 c 0.28 ± 0.04 b 0.20 ± 0.02 c 0.37 ± 0.02 a 0.33 ± 0.04 ab
mg/L)
Nitrite nitrogen (NO2 -N;
0.002 ± 0.000 ab 0.001 ± 0.000 b 0.002 ± 0.000 ab 0.001 ± 0.001 b 0.002 ± 0.001 b 0.003 ± 0.001 a
mg/L)
Nitrate nitrogen (NO3 -N;
0.28 ± 0.03 0.30 ± 0.02 0.32 ± 0.02 0.34 ± 0.03 0.30 ± 0.02 0.34 ± 0.03
mg/L)
Total nitrogen (TN; mg/L) 0.93 ± 0.18 d 1.02 ± 0.21 d 2.36 ± 0.61 c 1.22 ± 0.40 d 3.32 ± 0.32 b 7.03 ± 0.73 a
Total phosphorus (TP; mg/L) 0.12 ± 0.01 d 0.21 ± 0.04 c 0.25 ± 0.04 c 0.22 ± 0.04 c 0.34 ± 0.05 b 0.62 ± 0.04 a
pH 10.20 ± 0.30 10.15 ± 0.19 9.97 ± 0.50 10.38 ± 0.55 9.13 ± 0.59 10.37 ± 0.57
N/P ratio (NPR) 7.93 ± 1.01 bc 5.06 ± 1.83 c 9.30 ± 1.25 ab 5.96 ± 2.69 c 9.76 ± 0.56 ab 11.43 ± 1.87 a
3.2. Growth Parameters and Enzyme Activity

Group difference was found neither at day 0 nor at day 7 for all detected growth
parameters in E. sinensis (Table S1).
For the digestive enzymes, the CS7 group had slightly higher α-amylase activity (albeit
not significant) and significantly higher lipase activity than the CS0 group (Figure 1A,B).
At the same time, they had no significant difference between the NG0 and NG7 groups
or between the CN0 and CN7 groups. Interestingly, compared to the NG7 group, the
CN7 group had significantly lower digestive enzyme activities, but not the CS7 group
(Figure 1A,B).
For the antioxidant parameters, the NG7 group had slightly higher GR activity and sig-
nificantly higher SOD activity than the NG0 group (Figure 1C,D). Similarly, the CS7 group
had significantly higher GR activity and SOD activity than the CS0 group (Figure 1C,D).
At the same time, the CN7 group had slightly higher GR activity and similar SOD activity
than the CN0 group (Figure 1C,D). The NG7 group had slightly lower T-AOC activity than
the NG0 group, and the CS7 group had slightly higher T-AOC activity than the CS0 group,
while the CN7 group had similar T-AOC activity to the CS0 group (Figure 1E). Compared
to the respective initial groups, the NG7 and CS7 groups had similar MDA content, while
the CN7 group had slightly higher MDA content (Figure 1F). Interestingly, the level of
T-AOC and MDA in the CS7 group was more similar to the NG7 group than the CN7 group
(Figure 1E,F).
Figure 1. Variations in digestive enzymes activity and antioxidant parameters in the hepatopancreas
of Eriocheir sinensis across groups. (A) α-amylase activity; (B) lipase activity; (C) glutathione reductase
activity; (D) superoxide dismutase activity; (E) total antioxidant capacity; (F) malondialdehyde
content. Results are shown as mean ± SD (n = 6). Different letters indicate significant differences
among groups (p < 0.05).
3.3. General Analyses of High-Throughput Sequencing

In the 18 procaryotic libraries, raw reads ranged from 105,452 to 136,679, of which
99.80–99.88% were clean reads (Table S2A). While in the 18 eukaryotic libraries, raw reads
ranged from 120,136 to 136,857, of which 99.91–99.95% were clean reads (Table S2B).
In the 18 procaryotic libraries, the effective tags ranged from 94,675 to 124,686, the
effective ratio ranged from 86.70% to 92.90% (Table S2A), and the obtained OTUs ranged
from 619 to 1174 (Table S3A). While in the 18 eukaryotic libraries, the effective tags ranged
from 115,825 to 131,743, the effective ratio ranged from 96.15% to 97.54% (Table S2B), and
the obtained OTUs ranged from 259 to 561 (Table S3B).
For one given taxonomic classification level, the number of annotated effective tags
in procaryotic libraries varied a lot among samples, e.g., ranging from 77,861 to 113,368 at
the domain level and 4442–36,559 at the species level, respectively (Table 2A). Similarly,
the number of annotated effective tags in eukaryotic libraries varied a lot among samples,
e.g., ranging from 64,686 to 123,149 at the domain level and 3748–37,173 at the species level,
respectively (Table 2B).
To illustrate the characteristics of microbial and algal communities in different groups,

we classified all annotated taxa into three categories, i.e., bacteria, fungi, and eukaryotic
phytoplankton. The rarefaction curve of all samples supported the adequacy of the sam-
pling efforts (Figure S1). The rank abundance curves indicated the optimal richness and
evenness of bacteria and fungi in the NG groups, followed by the CN groups, and finally
the CS groups (Figure S2A,B). In addition, the NG7 group had an increased richness and
evenness of eukaryotic phytoplankton compared to the NG0 group, while the CS7/CN7
group had a decreased richness and evenness of eukaryotic phytoplankton compared to
the CS0/CN0 group (Figure S2C).
Table 2. The distribution of annotated tags in different taxonomic classifications.
Sequencing
Sample ID Domain Phylum Class Order Family Genus Species
Type
94,228 94,046 92,305 87,783 79,643
NG0-1 94,441 4442 (4.70%)
(99.77%) (99.58%) (97.74%) (92.95%) (84.33%)
109,473 109,278 107,170 99,595 90,443
NG0-2 109,676 6611 (6.03%)
(99.81%) (99.64%) (97.72%) (90.81%) (82.46%)
109,584 109,331 107,191 102,502 94,215 13,599
NG0-3 109,683
(99.91%) (99.68%) (97.73%) (93.45%) (85.90%) (12.40%)
104,428.33 104,218.33 102,222.00 96,626.67 88,100.33 8217.33
NG0 Mean 104,600.00
(99.84%) (99.64%) (97.73%) (92.38%) (84.23%) (7.86%)
113,250 113,179 111,854 109,463 93,563 16,892
NG7-1 113,368
(99.90%) (99.83%) (98.66%) (96.56%) (82.53%) (14.90%)
93,196 93,117 91,690 89,605 80,069 12,492
NG7-2 93,344
(99.84%) (99.76%) (98.23%) (95.99%) (85.78%) (13.38%)
105,065 105,038 104,181 102,587 91,716 14,042
NG7-3 105,136
(99.93%) (99.91%) (99.09%) (97.58%) (87.24%) (13.36%)
103,837.00 103,778.00 102,575.00 100,551.67 88,449.33 14,475.33
NG7 Mean 103,949.33
(99.89%) (99.84%) (98.68%) (96.73%) (85.09%) (13.93%)
80,075 80,059 79,755 79,382 77,371 18,650
CS0-1 80,161
(99.89%) (99.87%) (99.49%) (99.03%) (96.52%) (23.27%)
92,866 92,843 92,456 92,095 89,974 21,847
CS0-2 92,930
(99.93%) (99.91%) (99.49%) (99.10%) (96.82%) (23.51%)
101,201 101,183 100,785 100,202 97,461 24,437
CS0-3 101,269
(99.93%) (99.92%) (99.52%) (98.95%) (96.24%) (24.13%)
(A) 16S 91,380.67 91,361.67 90,998.67 90,559.67 88,268.67 21,644.67
CS0 Mean 91,453.33
rDNA (99.92%) (99.90%) (99.50%) (99.02%) (96.52%) (23.67%)
103,127 103,109 102,612 102,122 99,661 27,299
CS7-1 103,169
(99.96%) (99.94%) (99.46%) (98.99%) (96.60%) (26.46%)
89,763 89,723 89,283 88,832 87,016 15,630
CS7-2 89,880
(99.87%) (99.83%) (99.34%) (98.83%) (96.81%) (17.39%)
85,915 85,899 85,491 85,143 83,800 19,885
CS7-3 86,016
(99.88%) (99.86%) (99.39%) (98.99%) (97.42%) (23.12%)
92,935.00 92,910.33 92,462.00 92,032.33 90,159.00 20,938.00
CS7 Mean 93,021.67
(99.91%) (99.88%) (99.40%) (98.94%) (96.92%) (22.51%)
104,825 104,697 103,836 102,767 91,761 18,628
CN0-1 104,892
(99.94%) (99.81%) (98.99%) (97.97%) (87.48%) (17.76%)
89,740 89,601 88,652 87,415 76,336 20,547
CN0-2 89,884
(99.84%) (99.69%) (98.63%) (97.25%) (84.93%) (22.86%)
77,861 77,794 77,210 73,232 67,166 9676
CN0-3 78,021
(99.79%) (99.71%) (98.96%) (93.86%) (86.09%) (12.40%)
90,808.67 90,697.33 89,899.33 87,804.67 78,421.00 16,283.67
CN0 Mean 90,932.33
(99.86%) (99.74%) (98.86%) (96.56%) (86.24%) (17.91%)
100,312 100,238 99,440 95,114 87,891 15,071
CN7-1 100,515
(99.80%) (99.72%) (98.93%) (94.63%) (87.44%) (14.99%)
94,601 94,546 93,942 90,526 84,979 12,984
CN7-2 94,784
(99.81%) (99.75%) (99.11%) (95.51%) (89.66%) (13.70%)
105,597 105,113 102,437 98,134 73,850 36,559
CN7-3 105,671
(99.93%) (99.47%) (96.94%) (92.87%) (69.89%) (34.60%)
100,170.00 99,965.67 98,606.33 94,591.33 82,240.00 21,538.00
CN7 Mean 100,323.33
(99.85%) (99.64%) (98.29%) (94.29%) (81.97%) (21.47%)
Table 2. Cont.
Sequencing
Sample ID Domain Phylum Class Order Family Genus Species
Type
84,189 81,977 61,324 51,582 40,377
NG0-1 88,958 4435 (4.99%)
(94.64%) (92.15%) (68.94%) (57.98%) (45.39%)
103,956 102,627 87,267 47,380 38,737
NG0-2 108,113 4072 (3.77%)
(96.15%) (94.93%) (80.72%) (43.82%) (35.83%)
90,124 86,315 70,818 57,507 48,998
NG0-3 96,571 5936 (6.15%)
(93.32%) (89.38%) (73.33%) (59.55%) (50.74%)
92,756.33 90,306.33 73,136.33 52,156.33 42,704.00 4814.33
NG0 Mean 97,880.67
(94.76%) (92.26%) (74.72%) (53.29%) (43.63%) (4.92%)
83,440 75,461 74,327 66,750 36,212
NG7-1 92,084 5840 (6.34%)
(90.61%) (81.95%) (80.72%) (72.49%) (39.32%)
58,651 53,312 49,855 37,332 31,908
NG7-2 64,686 4782 (7.39%)
(90.67%) (82.42%) (77.07%) (57.71%) (49.33%)
65,141 60,398 56,825 45,686 31,030
NG7-3 71,318 4675 (6.56%)
(91.34%) (84.69%) (79.68%) (64.06%) (43.51%)
69,077.33 63,057.00 60,335.67 49,922.67 33,050.00 5099.00
NG7 Mean 76,029.33
(90.86%) (82.94%) (79.36%) (65.66%) (43.47%) (6.71%)
109,306 106,176 92,784 54,252 30,419 23,611
CS0-1 110,620
(98.81%) (95.98%) (83.88%) (49.04%) (27.50%) (21.34%)
121,756 118,267 99,510 54,048 38,118 28,296
CS0-2 122,808
(99.14%) (96.30%) (81.03%) (44.01%) (31.04%) (23.04%)
109,626 105,104 96,907 58,908 44,643 37,173
CS0-3 111,192
(98.59%) (94.52%) (87.15%) (52.98%) (40.15%) (33.43%)
(B) 18S 113,562.67 109,849.00 96,400.33 55,736.00 37,726.67 29,693.33
CS0 Mean 114,873.33
rDNA (98.86%) (95.63%) (83.92%) (48.52%) (32.84%) (25.85%)
116,239 115,048 104,639 68,167 31,416
CS7-1 120,699 7557 (6.26%)
(96.30%) (95.32%) (86.69%) (56.48%) (26.03%)
97,903 96,991 91,272 43,391 12,130
CS7-2 99,004 6774 (6.84%)
(98.89%) (97.97%) (92.19%) (43.83%) (12.25%)
102,140 100,721 90,683 48,204
CS7-3 104,438 8324 (7.97%) 3748 (3.59%)
(97.80%) (96.44%) (86.83%) (46.16%)
105,427 104,253 95,531 53,254 17,290
CS7 Mean 108,047.00 6026 (5.58%)
(97.58%) (96.49%) (88.42%) (49.29%) (16.00%)
118,061 114,076 104,969 67,345 39,861 13,201
CN0-1 119,050
(99.17%) (95.82%) (88.17%) (56.57%) (33.48%) (11.09%)
106,724 103,881 94,719 49,415 42,849 13,167
CN0-2 107,751
(99.05%) (96.41%) (87.91%) (45.86%) (39.77%) (12.22%)
73,221 70,057 62,733 53,005 45,577 16,810
CN0-3 77,136
(94.92%) (90.82%) (81.33%) (68.72%) (59.09%) (21.79%)
99,335 96,005 87,474 56,588 42,762 14,393
CN0 Mean 101,312.33
(98.05%) (94.76%) (86.34%) (55.86%) (42.21%) (14.21%)
56,933 54,757 48,899 35,657 28,831 13,392
CN7-1 59,490
(95.70%) (92.04%) (82.20%) (59.94%) (48.46%) (22.51%)
70,981 68,076 60,882 45,067 35,763 13,898
CN7-2 74,422
(95.38%) (91.47%) (81.81%) (60.56%) (48.05%) (18.67%)
121,951 119,134 109,838 64,912 58,258
CN7-3 123,149 8631 (7.01%)
(99.03%) (96.74%) (89.19%) (52.71%) (47.31%)
83,288 80,656 73,206 48,545 40,951 11,974
CN7 Mean 85,687.00
(97.20%) (94.13%) (85.43%) (56.65%) (47.79%) (13.97%)
3.4. Alpha and Beta Diversity

The Simpson diversity and Pielou’s evenness of bacteria had a significant difference
among groups, indicating the improved effect of supplementary EM and C. vulgaris on
the bacterial diversity and evenness of cyanobacterial bloom ponds (Table 3A). However,
the PD-whole tree index of fungi was significantly decreased in the CS7 group, indicat-
ing the inhibiting effect of supplementary EM and C. vulgaris on the fungal diversity of
cyanobacterial bloom ponds (Table 3B).
Interestingly, the Simpson diversity of eukaryotic phytoplankton was decreased in the
NG7 and CS7 groups than their initial levels, while it had no significant difference between
the CN0 and CN7 groups (Table 3C). Chao1, Ace, and PD-whole tree were decreased signif-
icantly in the CS7 group compared to the CS0 group. In contrast, these three indices had
different alterations in the NG7 and CN7 groups compared to their initial levels (Table 3C).
In eukaryotic phytoplankton, the number of species, OTU, and community diversity were
inhibited by a supplement of EM and C. vulgaris in cyanobacterial bloom ponds.
Table 3. Descriptive analysis and difference significance test of alpha diversity for each group.
Index NG0 NG7 CS0 CS7 CN0 CN7

Species richness 926.00 ± 79.38 813.67 ± 121.01 645.33 ± 18.48 666.33 ± 25.42 860.33 ± 269.31 961.67 ± 299.58
Shannon 5.58 ± 0.04 5.77 ± 0.03 4.23 ± 0.18 5.14 ± 0.26 5.14 ± 0.71 5.70 ± 0.46
Simpson 0.95 ± 0.00 a 0.95 ± 0.01 a 0.86 ± 0.02 c 0.93 ± 0.02 ab 0.90 ± 0.03 b 0.93 ± 0.02 ab
Chao1 1004.29 ± 123.04 893.14 ± 131.26 727.84 ± 32.90 738.00 ± 34.72 955.23 ± 268.91 1011.68 ± 320.48
(A) bacteria
Ace 998.00 ± 111.34 888.37 ± 131.95 739.90 ± 35.58 753.92 ± 39.43 968.22 ± 274.63 1021.39 ± 327.61
Good’s coverage
99.87 ± 0.05 99.87 ± 0.04 99.86 ± 0.02 99.87 ± 0.04 99.82 ± 0.06 99.88 ± 0.06
(×10−2 )
Pielou’s evenness 0.57 ± 0.01 ab 0.60 ± 0.01 a 0.45 ± 0.02 b 0.55 ± 0.03 ab 0.53 ± 0.05 ab 0.58 ± 0.02 a
PD-whole tree 116.50 ± 7.02 99.99 ± 16.24 86.19 ± 3.07 86.78 ± 4.42 106.76 ± 30.19 123.43 ± 32.08
Shannon 2.08 ± 0.30 2.51 ± 1.01 2.74 ± 0.42 2.52 ± 0.03 2.93 ± 0.90 3.33 ± 0.32
Simpson 0.52 ± 0.05 0.63 ± 0.27 0.72 ± 0.11 0.72 ± 0.01 0.76 ± 0.16 0.82 ± 0.04
Chao1 59.83 ± 8.58 71.73 ± 5.02 59.50 ± 13.54 37.82 ± 1.41 55.53 ± 21.37 65.01 ± 12.64
(B) fungi
Ace 59.69 ± 9.33 69.61 ± 8.96 61.42 ± 12.18 41.06 ± 1.15 55.39 ± 19.42 68.14 ± 8.04
Good’s coverage
99.87 ± 0.02 99.81 ± 0.07 99.83 ± 0.08 99.90 ± 0.02 99.86 ± 0.05 99.82 ± 0.05
(×10−2 )
Pielou’s evenness 0.36 ± 0.04 0.43 ± 0.17 0.48 ± 0.08 0.50 ± 0.00 0.52 ± 0.12 0.58 ± 0.04
PD-whole tree 5.63 ± 0.50 a 5.76 ± 0.14 a 5.10 ± 0.72 a 2.99 ± 0.29 b 4.78 ± 1.54 a 5.51 ± 1.10 a
Shannon 3.51 ± 0.17 bc 2.81 ± 0.09 c 4.69 ± 0.07 a 3.34 ± 0.37 abc 3.61 ± 0.13 b 3.78 ± 0.16 ab
Simpson 0.85 ± 0.02 ab 0.72 ± 0.02 c 0.92 ± 0.01 a 0.81 ± 0.07 bc 0.83 ± 0.03 bc 0.85 ± 0.01 ab
(C) phyto- Chao1 84.20 ± 6.42 b 100.20 ± 5.14 ab 101.68 ± 7.71 a 64.42 ± 12.37 c 98.71 ± 12.37 ab 104.77 ± 6.78 a
plankton Ace 84.03 ± 3.45 bc 101.71 ± 9.75 ab 102.69 ± 6.99 ab 65.86 ± 13.94 c 100.14 ± 13.96 ab 105.72 ± 13.79 a
Good’s coverage
99.89 ± 0.01 ab 99.85 ± 0.01 b 99.92 ± 0.01 a 99.92 ± 0.02 a 99.91 ± 0.02 a 99.88 ± 0.02 ab
(×10−2 )
Pielou’s evenness 0.57 ± 0.03 abc 0.44 ± 0.00 c 0.72 ± 0.01 a 0.57 ± 0.04 abc 0.55 ± 0.02 bc 0.58 ± 0.01 b
PD-whole tree 6.25 ± 0.40 b 7.41 ± 0.54 ab 7.53 ± 0.55 a 4.77 ± 0.62 c 7.48 ± 0.98 ab 7.36 ± 0.64 ab
Note: Different lowercase letters indicate significantly differences among groups.
PCoA plots revealed a separation of bacterial communities among groups regardless

of sampling time. Meanwhile, a high similarity existed between two sampling times
within a certain group (Figure 2A). As for the fungal communities, we can barely find
a difference among groups, but a separation existed between 0-day and 7-day of the CS
group (Figure 2B). A similar situation existed in the communities of phytoplankton, with
high similarity among groups regardless of the sampling time and separation between
samples of day 0 and day 7 in the CS or NG groups (Figure 2C). These results suggest
the microbiological compositions, especially the fungal and phytoplankton communities,
could be influenced by the supplement of EM and C. vulgaris.
Figure 2. PCoA plot base of the relative abundance of OTUs (97% similarity level) showing (A) bacte-
rial, (B) fungal and (C) phytoplankton structural clustering. Distance between samples was calculated
using the weighted unifrac method. ANOSIM was used to test the differences among groups.
3.5. Community Composition

In the NG groups, Proteobacteria, Firmicutes and Actinobacteria were the top 3 domi-
nant bacterial phyla (Figure 3A). Compared to the NG0 group, the relative abundance of
Firmicutes (NG0 = 17.11 ± 2.11, NG7 = 24.70 ± 2.98; Welch’s t-test p = 0.027) was increased,
while the relative abundance of Bacteroidetes (NG0 = 18.76 ± 1.17, NG7 = 9.31 ± 1.17;
Welch’s t-test p < 0.001) were decreased in the NG7 group. In contrast, the CS7 group
had lower relative abundance of Proteobacteria (CS0 = 54.93 ± 0.64, CS7 = 52.03 ± 1.28;
Welch’s t-test p = 0.040), and higher relative abundance of Bacteroidetes (CS0 = 1.41 ± 0.05,
CS7 = 11.49 ± 3.68; Welch’s t-test p = 0.042) than the CS0 group (Figure 3A). In addition,
compared to the CS0 group, the relative abundance of Cyanobacteria was decreased in the
CS7 group (CS0 = 1.26 ± 0.11, CS7 = 0.27 ± 0.10; Welch’s t-test p < 0.001). Interestingly,
compared to the similarity of community composition between the NG0 and CS0 groups,
higher similarity existed between the NG7 and CS7 groups (Figure 3A). There is no signifi-
cant difference in the relative abundance of bacterial phylum between the CN0 group and
the CN7 group.
Figure 3. Relative abundance of dominant (A) bacterial, (B) fungal and (C) phytoplankton phyla in
each group.
In the present study, the most abundant cyanobacterial genus was Microcystis PCC-
7914, accounting for 83.54% and 99.12% in the CS0 group and CN0 group, respectively
(Figure S3). In addition, genera of Cyanobium PCC-6307, Pseudanabaena PCC-7429, and
Leptolyngbya PCC-6306 were also detected in the cyanobacterial ponds (Figure S3).
The Ascomycota dominated the fungal community in all groups. Compared to the
CS0 group, the relative abundance of Chytridiomycota increased slightly in the CS7 group
(CS0 = 0.51 ± 0.09, CS7 = 2.02 ± 0.85; Welch’s t-test p = 0.098; Figure 3B). Interestingly, the
fungal community composition of the CS7 group converged with that of the NG7 group
and contrasted with that of the CN7 group (Figure 3B).
The phytoplankton community consisted of Chlorophyta, Streptophyta and Bacillario-
phyta phyla in all groups (Figure 3C). In the absence of exogenous influence, the NG group
had increased Bacillariophyta abundance at the 7-day sampling point (NG0 = 6.85 ± 1.81,
NG7 = 15.65 ± 5.09; Welch’s t-test p = 0.020). At the same time, the CN group had no
significantly altered phylum at the 7-day sampling point. In contrast, after the supplement
of EM and C. vulgaris, the relative abundance of Bacillariophyta was decreased significantly
in the CS group (CS0 = 28.51 ± 5.31, CS7 = 4.01 ± 1.10; Welch’s t-test p = 0.002; Figure 3C).
3.6. Microflora Alteration Associated with the Supplement of EM and C. vulgaris

Based on the relative abundance of OTUs, we used LEfSe to generate cladograms to
identify the specific microbial taxa associated with the supplement of EM and C. vulgaris
(Figures 4–6). We screened significantly enriched microbial abundances (LDA scores
(log 10) > 4.0) from the taxonomic phylum to species levels in the CS and CN groups. In
the CS group, we identified several significantly enriched taxa as key discriminants of
bacteria (Figure 4), fungi (Figure 5) and phytoplankton (Figure 6). However, only two
significantly enriched bacterial classes, Bacilli and Alphaproteobacteria, were screened as
key discriminants in the CN group (Figure S5).
Figure 4. Linear discriminant analysis (LDA) integrated with effect size (LEfSe). (A) Cladogram
indicating the phylogenetic distribution of bacterial taxa correlated with the CS0 or CS7 groups.
(B) The differences in abundance between the CS0 and CS7 groups.
indicating the phylogenetic distribution of fungal taxa correlated with the CS0 or CS7 groups.
In the CS7 group, Chryseobacterium (Bacteroidetes, Bacteroidia, Flavobacteriales, and

Weeksellaceae) was the most abundant bacterial genus (Figure 4B), while Weeksellaceae
was the most abundant bacterial family, followed by Rhizobiaceae (Proteobacteria, Al-
phaproteobacteria, Rhizobiales) and Enterobacteriaceae (Proteobacteria, Gammaproteobac-
teria, Enterobacteriales; Figure 4B). In the CS7 group, a 71.16-fold increase in genus
Chryseobacterium (Figure 4 and Figure S5A), as well as a 5.03-fold increase in family Rhizo-
biaceae and an 11.78-fold increase in family Enterobacteriaceae were observed compared to
the CS0 group (Figure 4 and Figure S6A).
indicating the phylogenetic distribution of phytoplankton taxa correlated with the CS0 or CS7 groups.
In the fungal taxa, the family Pleosporaceae (Ascomycota, Dothideomycetes, Pleospo-

rales) and the order Pleosporales were the most abundant fungal family and order in the
CS7 group, respectively (Figure 5B). In comparison, Hypocreales (Sordariomycetes) was
the most abundant fungal order in the CS0 group (Figure 5B). Compared to the CS0 group,
the CS7 group had a 33.41-fold increase in family Pleosporaceae, as well as a 3.36-fold
decrease in order Hypocreales (Figure 5, Figures S5B and S6B).
In the phytoplankton taxa, Mychonastes (Chlorophyta, Chlorophyceae, Sphaeropleales,
and Mychonastaceae) was the most abundant genus in the CS7 group, while Cyclotella
(Bacillariophyta, Coscinodiscophyceae, Thalassiosirales, Stephanodiscaceae) was the most
abundant genus in the CS0 group (Figure 6B). Compared to the CS0 group, the CS7 group
had a 3.33-fold increase in the genus Mychonastes and a decrease from 3.20 to 0 in the genus
Cyclotella (Figure 6, Figures S5C and S6C).
For those significantly altered microbial taxa in CS groups, we furtherly analyzed
the correlation between microbial taxa based on the relative abundance in all samples.
The fungal family Pleosporaceae was significantly negatively correlated with the phyto-
plankton family Stephanodiscaceae (rho = −0.609, p = 0.007; Table 4). Meanwhile, the
fungal order Hypocreales was significantly negatively correlated with the bacterial family
Burkholderiaceae (rho = −0.639, p = 0.004) and Chitinophagaceae (rho = −0.564, p = 0.015;
Table 4).
Table 4. Spearman correlation analysis based on the relative abundance of microbial taxa in each sample (n = 18). Upper diagonal data represent Spearman
correlation coefficients (rho). Lower diagonal data denote concomitant probabilities (p) of the two-tailed test.
Burkholderiaceae Microcystaceae Rhizobiaceae Chitinophagaceae Enterobacteriaceae Weeksellaceae Microscillaceae Pleosporaceae Hypocreales Dunaliellaceae Mychonastaceae Oocystaceae Stephanodiscaceae Pinnulariaceae
Burkholderiaceae −0.327 −0.059 0.948 * −0.077 −0.156 −0.440 0.032 −0.639 * 0.579 * −0.350 −0.478 * −0.309 −0.261
Microcystaceae 0.185 −0.364 −0.447 −0.389 −0.498 * 0.897 * 0.084 0.044 −0.564 * 0.513 * 0.622 * 0.037 0.205
Rhizobiaceae 0.817 0.137 −0.102 0.833 ** 0.839 * −0.515 * 0.015 −0.063 0.195 0.356 0.267 0.303 −0.443
Chitinophagaceae 0 0.063 0.687 −0.026 −0.189 −0.497 * −0.038 −0.564 * 0.628 * −0.410 −0.567 * −0.320 −0.276
Enterobacteriaceae 0.760 0.111 0 0.919 0.701 * −0.401 −0.209 0.032 0.079 0.428 0.294 0.199 −0.488 *
Weeksellaceae 0.537 0.035 0 0.453 0.001 −0.477 * 0.238 0.127 −0.044 0.179 −0.007 −0.027 −0.426
Microscillaceae 0.068 0 0.029 0.036 0.099 0.045 0.103 0.260 −0.754 * 0.461 0.476 * −0.176 0.152
Pleosporaceae 0.900 0.742 0.951 0.880 0.404 0.341 0.683 −0.453 −0.329 0.164 −0.267 −0.609 * −0.401
Hypocreales 0.004 0.861 0.804 0.015 0.900 0.616 0.297 0.059 −0.319 −0.098 0.16 0.267 0.389
Dunaliellaceae 0.012 0.015 0.438 0.005 0.754 0.861 0 0.182 0.197 −0.571 * −0.364 0.399 0.170
Mychonastaceae 0.155 0.030 0.147 0.091 0.076 0.478 0.054 0.515 0.699 0.013 0.690 * −0.035 −0.381
Oocystaceae 0.045 0.006 0.284 0.014 0.236 0.977 0.046 0.284 0.526 0.137 0.002 0.505 * −0.032
Stephanodiscaceae 0.212 0.883 0.222 0.195 0.429 0.917 0.486 0.007 0.284 0.101 0.890 0.033 0.394
Pinnulariaceae 0.295 0.414 0.066 0.268 0.040 0.078 0.548 0.099 0.111 0.499 0.119 0.900 0.105
Note: * indicates a significant correlation at 0.05 level, and ** indicates a significant correlation at 0.01 level.
To better understand the impacts of cyanobacterial blooms, we furtherly analyzed

the correlation between the most abundant genera of cyanobacteria and water quality
parameters, as well as physiological enzyme activities. Interestingly, Microcystis PCC-
7914 was significantly negatively correlated with T-AOC (rho = −0.943, p = 0.005) and
significantly positively correlated with MDA (rho = 0.943, p = 0.005), TAN (rho = 0.841,
p = 0.036), TN, TP, and NPR (rho = 0.943, p = 0.005; Table 5). In addition, Cyanobium PCC-6307
was significantly negatively correlated with AMS (rho = −0.943, p = 0.005; Table 5).
Table 5. Spearman correlation analysis based on the relative abundance of cyanobacterial genera in each group.
Microcystis PCC-7914 Cyanobium PCC-6307

rho p Value rho p Value
AMS −0.771 0.072 −0.943 0.005
LPS −0.771 0.072 −0.771 0.072
T-AOC −0.943 0.005 −0.771 0.072
SOD −0.486 0.329 −0.657 0.156
MDA 0.943 0.005 0.771 0.072
GR 0.200 0.704 0.029 0.957
TAN 0.841 0.036 0.58 0.228
NO2-N 0.772 0.072 0.494 0.32
NO3-N 0.530 0.280 0.441 0.381
TN 0.943 0.005 0.771 0.072
TP 0.943 0.005 0.771 0.072
pH −0.086 0.872 −0.429 0.397
NPR 0.943 0.005 0.600 0.208
3.7. Functional Alteration Associated with the Supplement of EM and C. vulgaris

We applied two different approaches, FAPROTAX and Bugbase, to predict the eco-
logical functions of bacterial communities. Compared to the CS0 group, the CS7 group
had enhanced function of chemoheterotrophy, accompanied by inhibited functions of hu-
man_pathogens_all and human_pathogens_nosocomia (Figure 7A and Table S4A). Besides
the abovementioned functions, several metabolic phenotypes were associated with the sup-
plement of EM and C. vulgaris. Compared to the CS0 group, the CS7 group had increased
phenotypes of Gram_Negative and Facultatively_Anaerobic, accompanied by decreased
phenotypes of Aerobic and Gram_Positive (Figure 7B and Table S4B).
Figure 7. Variations in metabolic phenotypes and ecological functions of prokaryotes in E. sinensis pond.
(A) top 10 functions annotated using FAPROTAX and (B) phenotypic abundance analyzed with Bugbase.
4. Discussion
4.1. Impact of Cyanobacterial Bloom on E. sinensis Culture Pond
Cyanobacterial blooms can cause problems with water quality, alter the bacterial
community assembly, disturb the physiological function and impair the health of aquatic
animals [6,9,35]. Thus, cyanobacterial blooms are often disasters for aquatic ecosystems
and their living organisms. In the present study, the most abundant cyanobacterial genus
was Microcystis PCC-7914 in cyanobacterial bloom ponds, and the NPR was low (<11.43).
These results were compatible with the viewpoint that non-N2-fixing Microcystis tended
to dominate cyanobacterial blooms when N became limited (low NPR) [15]. Congruent
with the close relationship between N, P nutrients and cyanobacterial bloom [36], in the
present study, we found Microcystis PCC-7914 was significantly correlated with TAN, TN,
TP and NPR. Thus, we suggested cyanobacterial blooms degraded the water quality of the
E. sinensis culture pond by increasing the content of TAN, TN and TP.
Exposure to Microcystis spp. altered antioxidant enzymes activities in fish [37–39] and
crustaceans (e.g., Pacific white shrimp Litopenaeus vannamei) [35], as well as the digestive
enzymes activity in rotifer (Brachionus calyciflorus) [40]. Similarly, in the present study,
E. sinensis from the cyanobacterial bloom ponds had decreased digestive enzyme activ-
ity and total antioxidant capacity than that from the normal ponds. Antioxidant sys-
tems were efficient defenders of organisms against the negative impacts of toxicants. In
E. sinensis, short-term acute toxicity of MC-LR (within 48 h) often caused enhanced antioxi-
dant ability [5,24]. Contrast to that, we found prolonged exposure (7 d) to Microcystis spp.
decreased digestive enzymes activity and total antioxidant capacity of E. sinensis. The
dysfunction of digestive and antioxidant system in E. sinensis confirmed the Microcystis spp.
disturbed the physiological function of aquatic animals once again.
According to the study in eutrophic lake sediments, Firmicutes is a major phylum with
high global centrality in the microbial network; its relative abundance is close connections
with the cyanobacterial blooms phase [41]. In the present study, cyanobacterial blooms
decreased the relative abundance of Firmicutes and increased the relative abundance of
Cyanobacteria. A similar result was found in the previous study that MC-LR exposure de-
creased the relative abundance of Firmicutes in the intestinal microbiota of L. vannamei [36].
In addition, cyanobacterial blooms altered the community assembly of fungal and phyto-
plankton in the E. sinensis culture pond, representing the decreased relative abundance of
Ascomytcota and Streptophyta and the increased relative abundance of Chytridiomycota
and Chlorophyta.
4.2. The Supplement of EM and C. vulgaris Improved Water Quality of E. sinensis Culture Pond
An aquaculture pond is a complex ecosystem consisting of different types of organisms
interacting with each other as well as with the surrounding environmental conditions. The
organisms in the culture pond of E. sinensis are diverse, including aquatic plants, algae,
zooplankton, crabs, mud snails, and microorganisms. The stability of the pond ecosystem is
the key to the success of aquaculture, which is strongly associated with biotic components
and abiotic factors. In aquaculture, water quality parameters (e.g., dissolved oxygen, pH,
nitrogen and phosphorus nutrients) are commonly used abiotic indicators for monitoring
the health of the pond ecosystem [42,43]. In the present study, a supplement of EM and
C. vulgaris decreased TAN and TN levels, confirming their improved effect on the water
quality of cyanobacterial bloom ponds.
Since many species of cyanobacteria had outstanding abilities in nitrogen fixation [44,45],
even low NPR favored blooms of nitrogen-fixing cyanobacteria [46]. In the present study,
NPR was significantly decreased after the supplement of EM and C. vulgaris in cyanobacte-
rial bloom ponds, while it had a similar slight decrease in normal ponds. In contrast, NPR
was slightly increased, accompanied by the increase of cyanobacteria in cyanobacterial
bloom ponds without supplements. Rather than cyanobacteria [47], our results indicated
that the blooms of other algae are more relevant to the decrease of NPR. Interestingly, in
line with the previous study [48], our results favored a positive correlation between NPR
and cyanobacteria abundance.
In the present study, these water quality parameters were all in good condition ex-
cept for the total nitrogen and total phosphorus in several cyanobacterial blooms ponds,
suggesting a certain but limited role of water quality parameters on risk warning for the
pond ecosystem.
4.3. Effect of the Supplement of EM and C. vulgaris on the Growth and Physiology of E. sinensis
Similar to the previous study [4], in the present study, we found no significant effect
of the supplement of EM and C. vulgaris on the growth of female E. sinensis. Other than the
limited effect of splashing EM into the water, in this study, the lack of change in the growth
parameters of E. sinensis should be mainly attributed to the short experiment period.
In the present study, the digestive enzyme activity of E. sinensis, especially the LPS,
was increased by the supplement of EM and C. vulgaris. Similarly, antioxidant enzymes
(GR and SOD), as well as the total antioxidant capacity, were increased by the supplement
of EM and C. vulgaris. Meanwhile, the constant level of MDA in the CS group but not in
the CN group indicates the inhibition of lipid peroxidation in the hepatopancreas of crab
by the supplement of EM and C. vulgaris [49–51]. Thus, we suggested the supplement of
EM and C. vulgaris into the water improved the digestive and antioxidant ability of female
E. sinensis.
4.4. Bacterial Compositions Were Restored by the Supplement of EM and C. vulgaris

The genus Chryseobacterium is classified within the family Weeksellaceae, order Flavobac-
teriales, class Bacteroidia, and in the phylum Bacteroidetes. Chryseobacterium species were
previously classified in the genus Flavobacterium. Chryseobacterium species are rare op-
portunistic pathogens of low virulence, and their presence in clinical specimens usually
represents colonization and not infection [52,53]. Thus, the dominant abundance of the
Chryseobacterium species in the CS7 group suggested they captured more space and nutri-
ents than other species. The family Rhizobiaceae is classified within the order Rhizobiales,
class Alphaproteobacteria, and in the phylum Proteobacteria. Rhizobiaceae can fix atmo-
spheric nitrogen, which is a process that requires a lot of energy and electrons [54]. Thus,
Rhizobiaceae seems a competitive inhibitor for nitrogen-fixing cyanobacteria. Given the
decrease of the phylum Cyanobacteria along with the increase of the family Rhizobiaceae
in the present study, as with the previous study [55], we suggest the prospect of employ-
ing Rhizobiaceae in bioremediation is promising. Enterobacteriaceae is a family of the
order Enterobacteriales, class Gammaproteobacteria, and in the phylum Proteobacteria.
Enterobacteriaceae is present in various niches worldwide [56], while most members of
Enterobacteriaceae are opportunistic pathogens for aquatic animals [57]. At the same time,
some Enterobacter species provide significant protection against Flavobacterium psychrophilum
infection in Oncorhynchus mykiss [58–60].
In the present study, we found a 1.06-fold decrease in the abundance of phylum Pro-
teobacteria and a 4.67-fold decrease in Cyanobacteria associated with the supplement of
EM and C. vulgaris, along with the increase of the genus Chryseobacterium (71.16-fold), the
family Rhizobiaceae (5.03-fold) and the family Enterobacteriaceae (11.78-fold), represent-
ing a more space and nutrients colonized by neutral or beneficial bacteria than harmful
species [23,61]. Interestingly, the supplement of EM and C. vulgaris were predicted to
enhance the function of chemoheterotrophy and inhibit functions of human_pathogens_all
and human_pathogens_nosocomia. Together with the higher similarity in the community
composition of the NG7 and CS7 groups in the present study, we verified the bacterial
compositions of cyanobacterial blooms ponds could be restored by the supplement of EM
and C. vulgaris.
4.5. Fungal Roles in the Ecosystem of E. sinensis Culture Pond

In the present study, the fungal community composition of the CS7 group converged
with that of the NG7 group and contrasted with that of the CN7 group, suggesting the
supplement of EM and C. vulgaris promoted the restoration of the fungal community
composition in cyanobacterial blooms ponds.
In the present study, we found a 33.41-fold increase in the family Pleosporaceae, a
3.96-fold increase in the phylum Chytridiomycota, as well as a 3.36-fold decrease in the
order Hypocreales, which were significantly associated with the supplement of EM and
C. vulgaris. Chytridiomycota, commonly called chytrids, are a phylum of fungi in which
most species live in aquatic environments, and some species are also abundant in soil. In
aquatic environments, Chytridiomycota mostly forms scanty filaments with sporangia [62].
As saprophytes, they can degrade chitin and cellulose [63], thus playing a very important
role as decomposers in ecosystems. Pleosporaceae is a family in the order Pleosporales,
the class Dothideomycetes, and the phylum Ascomycota. While the order Hypocreales
is within the class Sordariomycetes, the phylum Ascomycota. Most of the members in
the family Pleosporaceae and the order Hypocreales are opportunistically pathogenic
species for vertebrates [64]. A large portion of Hypocreales fungi is mycoparasitic or
mycosaprotrophic [65], and several Hypocreales fungi have been used for controlling
spider mites [66].
Interestingly, in the present study, we found a significantly negative correlation be-
tween the fungal family Pleosporaceae and the phytoplankton family Stephanodiscaceae,
and so does the fungal order Hypocreales with the bacterial family Burkholderiaceae
and Chitinophagaceae. Our findings suggested Pleosporaceae may be pathogenic fungi
for Stephanodiscaceae (Bacillariophyta) or the metabolites of Pleosporaceae inhibited the
growth and reproduction of Stephanodiscaceae. Meanwhile, Hypocreales fungi may be
competitive inhabitors for Burkholderiaceae and Chitinophagaceae bacteria.
4.6. The Supplement of EM and C. vulgaris Altered Phytoplankton Compositions of E. sinensis

Culture Pond
The genus Mychonastes belongs to the family Mychonastaceae, the order Sphaero-
pleales, the class Chlorophyta, and the phylum Chlorophyceae. It is difficult to distinguish
Mychonastes algae from morphology alone for their lack of distinctive morphological fea-
tures [67]. Based on the phylogenetic marker, Krienitz et al. (2011) [68] confirmed the
synonymy of Mychonastes homosphaera and Chlorella minutissima and furtherly proposed
a taxonomic revision of the genus Mychonastes. The genus Mychonastes widely exists in
inland waters, which comprise spherical, ovoid or ellipsoidal algae living solitarily or in
small groups [68]. Interestingly, M. homosphaera seems to be a high-quality food source for
zooplankton and heterotrophic flagellates and also a promising feedstock for high-quality
animal feed [69]. In the present study, the abundance of the genus Mychonastes was in-
creased after the supplement of EM and C. vulgaris, providing us with a safe and efficient
method for potentially fertilizing aquaculture ponds.
Similar to the Mychonastes dominating the photosynthetic picoeukaryotes in Lake
Poyang [70], we found Mychonastes was representative of the prominent phytoplankton in
the E. sinensis culture pond ecosystem. Since the ponds were connected with the Yangtze
River, in the present study, we confirmed the representativeness of the Mychonastes species
in river-connected aquatic environments [70].
The genus Cyclotella belongs to the family Stephanodiscaceae, the order Thalassiosir-
ales, the class Coscinodiscophyceae, and the phylum Bacillariophyta. Most Cyclotella algae
live in freshwater environments, and very rare species live in saline water [71–73]. Amongst
these, Cyclotella meneghiniana is an ideal feed for the larvae of E. sinensis [74]. Particularly,
the ability to accumulate lipids for energy storage makes Cyclotella diatoms industrially
attractive for the production of biofuels and high-value compounds [75,76]. In diatoms,
Cyclotella is a better competitor for silicate, while Asterionella is a better competitor for
phosphorus [77]. In the present study, the decrease of the genus Cyclotella (Bacillariophyta)
and the increase of the genus Mychonastes (Chlorophyta) in the CS7 group suggested that
Mychonastes rather than Cyclotella is a better competitor for nitrogen.
4.7. The Application Prospect of Probiotics and Microalgae in Aquaculture

It is well known that the presence of cyanotoxins and water pollution could cause
undesirable chemicals to become residual, especially in the hepatopancreas (the main edible
part), thereby reducing the quality of E. sinensis. Therefore, developing more sustainable
models of E. sinensis breeding is critical. Given the interactions between microalgae and
bacteria [78], it is an applicable way to regulate the pond ecosystem with a combination
of probiotics and microalgae in aquaculture [22,79]. In the present study, we found no
adverse effects of EM and C. vulgaris supplementation in the water on the growth, digestive
or antioxidant ability of E. sinensis. Specifically, EM and C. vulgaris supplementation
counteracted potential problems caused by cyanobacterial blooms. To prevent and control
the occurrence of cyanobacterial blooms in aquaculture, we suggested using a combination
of EM and C. vulgaris to maintain a stable pond ecosystem as early as possible.
5. Conclusions
In the present study, we found no adverse effects of EM and C. vulgaris supplementa-
tion in the water on the growth, digestive or antioxidant ability of E. sinensis. We assessed
the potential combined effects of EM and C. vulgaris on the prevention and control of
cyanobacteria in E. sinensis culture ponds. As a result, the supplement of EM and C. vulgaris
improved the water quality of the E. sinensis culture pond by reducing TAN and TN levels.
We found an improved effect on the bacterial diversity and evenness while inhibiting effects
on the diversity of fungal and phytoplankton communities were related to the supple-
ment of EM and C. vulgaris. Meanwhile, supplementary EM and C. vulgaris had complex
effects on the microbial compositions in the E. sinensis pond. In bacterial compositions,
the supplement of EM and C. vulgaris increased the abundance of Gram-negative bacteria
and facultatively anaerobic species, whereas it decreased the abundance of aerobic and
Gram-positive bacteria. Based on FAPROTAX, we predicted the enhanced function of
chemoheterotrophy, accompanied by inhibited functions of human_pathogens_all and
human_pathogens_nosocomia, were associated with the supplement of EM and C. vulgaris.
Interestingly, the supplement of EM and C. vulgaris promoted the restoration of the bacterial
and fungal community composition in cyanobacterial blooms ponds. In phytoplankton,
the supplement of EM and C. vulgaris increased the abundance of the genus Mychonastes
(Chlorophyta) while decreasing that of the genus Cyclotella (Bacillariophyta). In addition,
we found a significantly negative correlation between the fungal family Pleosporaceae and
the phytoplankton family Stephanodiscaceae, and so does the fungal order Hypocreales
with the bacterial family Burkholderiaceae and Chitinophagaceae. The present study is a
practice of regulating the pond ecosystem with a combination of probiotics and microalgae.
Our findings laid the foundation for the prevention and control of potential risks in the
culture of E. sinensis.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/su15097362/s1, Figure S1: Rarefaction curve evaluating the
relative richness of (A) bacteria, (B) fungi and (C) phytoplankton; Figure S2: OTU rank abun-
dance curve to visualize evenness and richness of (A) bacterial, (B) fungal and (C) phytoplankton
species; Figure S3: Relative abundance of the most abundant cyanobacterial genus in each group;
Figure S4: Linear discriminant analysis (LDA) integrated with effect size (LEfSe). (A) Cladogram in-
dicating the phylogenetic distribution of phytoplankton taxa correlated with the CN0 or CN7 groups.
(B) The differences in abundance between the CN0 and CN7 group; Figure S5: Microbial taxa with
significant abundance differences between the CS0 and CS7 groups. (A) different bacterial genera,
(B) different fungal families, (C) different genera of phytoplankton. Statistical differences were as-
sessed using Welch’s t-tests; Figure S6: Microbial taxa with significant abundance differences between
the CS0 and CS7 groups. (A) different bacterial families, (B) different fungal order, (C) different fami-
lies of phytoplankton. Statistical differences were assessed using Welch’s t-tests; Table S1: Description
statistic of the growth parameters for female crabs. Values are presented as means (±SD) of three
pond replicates; Table S2: Basic information of 18 procaryotic and 18 eukaryotic sequencing libraries;
Table S3: The number of tags and OUTs of 18 procaryotic and 18 eukaryotic sequencing libraries;
Table S4: Descriptive statistics for the relative abundance of (A) ecological functions and (B) metabolic
phenotypes of prokaryotes in E. sinensis pond. (A) top 10 functions annotated using FAPROTAX and
(B) phenotypic abundance analyzed with Bugbase. Significant differences between 0-day and 7-day
sampling time within a group were indicated by an asterisk.
Author Contributions: Conceptualization, Writing-Original draft preparation & Funding acquisition,
J.G. and G.X.; Visualization & Investigation, J.G., N.S. and Y.S.; Data curation, Z.N. and X.Y.; Resources,
X.Y. and F.D.; Funding acquisition, Supervision &Writing-review & editing, G.X. and P.X. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the “JBGS” Project of Seed Industry Revitalization in Jiangsu
Province (grant number JBGS [2021]125) and the earmarked fund for CARS48.
Institutional Review Board Statement: The animal study protocol was approved by the Ethics
Committee of Nanjing Agricultural University (protocol code SYXK (Xu) 20210701 and 1 July 2021).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are openly available at https://www.
ncbi.nlm.nih.gov/sra/?term=PRJNA835239 (accessed on 1 June 2022).
Acknowledgments: We are indebted to all reviewers and editors for their contributions to this paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Impact of Effective Microorganisms and Chlorella Vulgaris On

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sustainability

Sustainability 2023, 15, 7362. https://doi.org/10.3390/su15097362 https://www.mdpi.com/journal/sustainability

2. Materials and Methods

2.2. Sampling and Water Quality Detection

2.3. Enzyme Activity and Antioxidant Parameters Assays

2.4. DNA Extraction, PCR Amplification, and Sequencing

2.5. Tags Assembly, Filtering, and Taxonomy Annotation

2.6. Community Composition and Differentially Abundant Taxons

2.7. Alpha and Beta Diversity Analysis

2.8. Statistical Analysis

Water Quality Parameters NG0 NG7 CS0 CS7 CN0 CN7

3.2. Growth Parameters and Enzyme Activity

3.3. General Analyses of High-Throughput Sequencing

To illustrate the characteristics of microbial and algal communities in different groups,

Table 2. The distribution of annotated tags in different taxonomic classifications.

3.4. Alpha and Beta Diversity

Index NG0 NG7 CS0 CS7 CN0 CN7

PCoA plots revealed a separation of bacterial communities among groups regardless

3.5. Community Composition

3.6. Microflora Alteration Associated with the Supplement of EM and C. vulgaris

In the CS7 group, Chryseobacterium (Bacteroidetes, Bacteroidia, Flavobacteriales, and

In the fungal taxa, the family Pleosporaceae (Ascomycota, Dothideomycetes, Pleospo-

To better understand the impacts of cyanobacterial blooms, we furtherly analyzed

Microcystis PCC-7914 Cyanobium PCC-6307

3.7. Functional Alteration Associated with the Supplement of EM and C. vulgaris

4.4. Bacterial Compositions Were Restored by the Supplement of EM and C. vulgaris

4.5. Fungal Roles in the Ecosystem of E. sinensis Culture Pond

4.6. The Supplement of EM and C. vulgaris Altered Phytoplankton Compositions of E. sinensis

4.7. The Application Prospect of Probiotics and Microalgae in Aquaculture

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