Aquaculture 554 (2022) 738057

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Enhancing effect of Platymonas addition on water quality, microbial

community diversity and shrimp performance in biofloc-based tanks for
Penaeus vannamei nursery
Sheng Dong a, Yu Li b, Fei Huang b, Langli Lin b, Ze Li b, Jiancheng Li c, Yuxiu Zhang a, *,
Yihong Zheng b, *
a
School of Chemical and Environmental Engineering, China University of Mining and Technology (Beijing), Beijing 100083, China
b
Guangdong Provincial Key Laboratory for Plant Epigenetics, Guangdong Engineering Research Center for Marine Algal Biotechnology, College of Life Sciences and
Oceanography, Shenzhen University, Shenzhen 518060, China
c
Instrumental Analysis Center, Shenzhen University, Shenzhen 518060, China

A R T I C L E I N F O A B S T R A C T

Keywords: Biofloc technology (BFT) works by stimulating a microbial community of different metabolic types in order to
Biofloc technology simultaneously treat pollutants in situ while improving aquaculture. Although microalgae are important auto­
Microalgal enhancement trophic microorganisms in aquaculture systems, their role in BFT is rarely explored. In this study, Platymonas sp.
Microflora
was selected as a bioaugmentation microalgae for Penaeus vannamei nursery in a BFT system. The experimental
Antioxidant capacity
Growth performance
group with microalgae additions to 1 × 105 cell⋅mL− 1 every 4 days, were compared to the control group without
microalgae addition based on the water quality, biofloc community structure, and enzyme activity measure­
ments. The results showed that the bioaugmentation of Platymonas sp. reduced nitrite‑nitrogen accumulation in
rearing water and improved alpha diversity of bacterial communities both in shrimp intestines and in biofloc.
Platymonas sp. may improve the microbiological properties of bioflocs by increasing the relative abundance of
beneficial bacteria in shrimp aquaculture including Prosthecobacter, Denitromonas, Rheinheimera, Mycobacterium,
Roseobacter, Phaeodactylibacter, Cellvibrio and Nannocystis, which help promote nitrification, denitrification, and
formation of bioflocs. After 30-day rearing, zootechnical performances of shrimps from the experimental group
were significantly higher than those of the control group, including survival rates, specific growth rates and
weight gains per week of shrimp each increasing by 63.3%, 7.6% and 53.0%, respectively. Furthermore,
microalgal bioaugmentation improved the hepatopancreatic antioxidant status of P. vannamei by significantly
increasing the activity of the antioxidant enzymes superoxide dismutase, catalase, and peroxidase in the
experimental group. Overall, the results of this study demonstrated that Platymonas sp. addition could simul­
taneously improving rearing environment and nursery performance for P. vannamei in a biofloc-based system,
indicating the important role of microalgae on the BFT application for shrimp nursery.

1. Introduction
The aquaculture industry plays a crucial role in agriculture, espe­
cially since the rapid growth of Penaeus vannamei farming in recent
years. Notably, the effluent pollution of the conventional shrimp
farming model is causing increasing concern, due to high-density
farming, excess feed consumption, and intensive water exchange. Bio­
floc technology (BFT), a low-cost, environment-friendly, and convenient
aquaculture strategy has received extensive attention in recent years,
especially in the field of P. vannamei aquaculture (El-sayed, 2020; Ray,
2012). A major advantage of the BFT system over conventional shrimp
farming is that BFT is an in-situ treatment process for wastewater and
can therefore ensure the quality of both rearing water and effluent. Since
BFT does not require the construction of multiple external equipment or
filtration systems, start-up investment and operating costs are reduced
significantly. Secondly, bioflocs can be used as living foods for shrimp
and other aquaculture animals to improve feed efficiency and provide
extra nutrition (Anand et al., 2014; Ray, 2012; Tibbetts et al., 2017).
As a complex aquatic microcosm, biofloc consists of microorganisms
with different ecological niches and metabolic profiles (Ferreira et al.,

* Corresponding authors.
E-mail addresses: zhangyuxiu@cumtb.edu.cn (Y. Zhang), zhengy@szu.edu.cn (Y. Zheng).

https://doi.org/10.1016/j.aquaculture.2022.738057
Received 17 November 2021; Received in revised form 13 February 2022; Accepted 16 February 2022
Available online 19 February 2022
0044-8486/© 2022 Published by Elsevier B.V.
S. Dong et al.
2015). Among them, autotrophic microorganisms include microalgae,
photosynthetic bacteria, and chemoautotrophic bacteria (Robles-
Porchas et al., 2020). While heterotrophic bacteria can degrade residues
by consuming a variety of organic carbon sources, which are then con­
verted into bacterial biomass. In BFT system, zooplankton typically
include protozoa such as ciliates, flagellates and sarcodinas, as well as
metazoa such as rotifers and nematodes. As food organisms of shrimp,
zooplanktons, which usually feed on bacteria, algae, residual feed, and
feces, are critical to nutrient transfer (Ferreira et al., 2015).
The profound value of microalgae in BFT is always overlooked. Aside
from assimilating pollutants such as inorganic nitrogen and phosphorus,
microalgae can increase organic carbon content in rearing water and
provide organic substances for heterotrophic microorganisms in bio­
flocs. This further improves the aquaculture environment (Gupta et al.,
2019; Llario et al., 2018; Nie et al., 2020; Yang et al., 2020). Also,
microalgae can introduce a variety of antioxidants, including poly­
unsaturated fatty acids, photosynthetic pigments and carotenoids,
phenolic compounds, metabolic intermediates, phycobiliproteins,
sulfated lipids and polysaccharides, and vitamins to aquatic animals
(Chen et al., 2021; Shalaby, 2011). They may help reduce reactive ox­
ygen species (ROS) in shrimp under stressful aquaculture conditions (Ge
et al., 2016; Rezayian et al., 2019; Zheng et al., 2020). Active com­
pounds are carried through the food chain and accumulate in cultured
animals, which can reduce oxidant stress effects, improve antioxidant
properties, and ultimately improve shrimp production. (Anaya-Rosas
et al., 2019; Kusmayadi et al., 2021; Tibbetts et al., 2017; Tiong et al.,
2020). In addition, shrimp accumulate pigments in made by algae (Lu
et al., 2021; Nickell and Bromage, 1998), which affects the body color of
shrimp and their commercial value (Giang et al., 2019). Thus, the role of
microalgae in BFT is worthy of further exploration.
In aquaculture, or in wastewater treatment, microalgal organisms
such as Chlorella, Phaeodactylum, Scenedesmus, and Nannochloropsis are
regularly studied (Jung et al., 2017; Llario et al., 2018; Ni et al., 2018;
Nie et al., 2020). A recent study confirmed the positive role of Nanno­
chloropsis oculata and Thalassiosira pseudonana were benificial to water
quality and development of P. vannamei aquaculture (Huang et al.,
2022). The effect of microalgae Schizochytrium sp. on water quality and
growth performance of P. vannamei in BFT tanks was reported (Pacheco-
Vega et al., 2018). In Tetraselmis genus, Platymonas are free-swimming
green algae rich in zeaxanthin and polyunsaturated fatty acids (Huang
and Cheung, 2011; Selvakumar and Umadevi, 2014; Huang et al.,
2020a), making it a popular source of feed for aquatic hatchlings to
increase survival rates and resistance to pathogens (Ge et al., 2016; Ge
et al., 2017). Furthermore, Platymonas with its excellent inorganic ni­
trogen removal ability may benefit to the control of water quality in BFT
systems (Dong et al., 2021; Guo et al., 2013). However, due to the
accumulation of feeding, fecal wastes, microorganisms and bioflocs, the
increase of turbidity in BFT system can cause the demise or even absence
of microalgae because of the limitation of light, especially in the middle
and late stage of shrimp farming (Reis et al., 2019). Bioaugmentation is a
promising technique for solving performance and efficiency problems
during wastewater bioremediation process by adding selected functional
microorganisms, especially pure bacterial strains which have been
extensively applied to improve the degradation efficiency of toxic
recalcitrant compoundsfrom refractory wastewater (Tale et al., 2015).
However, studies on the bioaugmentation application of microalgae in
BFT-based aquaculture are still limited. The current study evaluated the
bioaugmentation effects of Platymonas microalge on water quality pa­
rameters, bacterial community composition, zootechnical performance
and antioxidant status of P. vannamei raised in an indoor BFT system.
2. Materials and methods
2.1. Algal species, shrimp and bioflocs
In this experiment, the unicellular microalgae Platymonas sp.
2
Aquaculture 554 (2022) 738057
(CCMA-308) was obtained from the Center for Collections of Marine
Algae (CCMA, Xiamen, China). Platymonas sp. was inoculated in steril­
ized f/2 liquid medium of 10 g⋅L− 1 salinity and incubated in a lighted
oscillating incubator with shaking speed of 120 rotation per minute at
25 ± 2 ◦ C temperature, under a 12:12 light-dark cycle with a light in­
tensity of 180 μE photons m− 2 s− 1 to logarithmic stage.
To promote shrimp growth and match the most actual situation of
shrimp offshore farming, the salinity of 10 g⋅L− 1 was selected as the
culture condition. Specific pathogen free (SPF) P. vannamei post larvae
(PL10) reared at a salinity of 10 g⋅L− 1 were obtained from Shenwan
Zhengda shrimp hatchery (Zhongshan, China). After an acclimatization
of 48 h in 10 g⋅L− 1 salinity brackish water, P. vannamei in good condition
and uniform length were used for experiment.
The bioflocs used in this experiment were cultivated by the labora­
tory. Brackish water with a salinity of 10 g⋅L− 1 was prepared using fresh
seawater and tap water and disinfected. Bioflocs were cultivated using
commercial compound EM probiotic concentrated powder (1 g⋅100 L− 1
brackish water, Nanhua Qianmu, China) which was resuspended over­
night according to the instructions and then inoculated proportionally at
12:00 each day for 3 d. After that, the culture was continuously sup­
plemented with sucrose (4.75 mg/L) and NH4Cl (0.76 mg/L) at a rate of
C/N of 10:1 for 7 days refer to previous reports (Avnimelech, 1999;
Ebeling et al., 2006; Ray et al., 2011), and fine flocs were formed with
SV30 of about 1–2‰ using Imhoff settling cone. When nitrite‑nitrogen
(NO2-N) and total ammonia nitrogen (TAN) in water were less than 0.2
mg⋅L− 1 for three consecutive days, the cultivation of bioflocs were
completed.
2.2. Experimental grouping and shrimp rearing
The study consisted of a microalgal addition treatment (MA) and a
control group (CK) both in three replicates, which were performed in
white buckets with 50 L mature bioflocs each. For the microalgal
addition treatment, freshly cultured Platymonas sp. was inoculated
initially and enhanced every four days at a density of 1 × 105 cell⋅mL− 1
with reference to former reports (Ge et al., 2017; Dong et al., 2021).
Shrimp of 100 PL12 described in 2.1 were added to each bucket. Before
rearing, the initial mean body weight of P. vannamei at PL12 was
measured and calculated using extra shrimps. During a 30-day period,
both groups were fed by hand four times per day at fixed intervals. For
the first 10 days, the shrimps were fed based on 20% of the stocking
biomass with initial feed (crude protein content 48%, Tong Wei, China).
The daily feed ration was decreased to 15% of the assumed shrimp
biomass with crushed feed (particle size 1 mm, crude protein content
40%, Tong Wei, China) from day 11 to 30, and the rations were adjusted
daily according to the observation of feeding trays (Xu et al., 2020).
Meanwhile, additional sucrose was replenished to achieve an input
organic carbon (including feed carbon) to input organic nitrogen ratio
(C/N) of 12 (Avnimelech, 1999; Ebeling et al., 2006; Ray et al., 2011).
2.3. Determination of water quality parameters
During the experimental period, the physicochemical parameters of
all buckets were monitored. Water from both groups was sampled before
the first feeding every day. After filtered through a 0.45 μm pore size
membrane, TAN, NO2-N, and total alkalinity were analyzed by Clever
Chem 380 automatic water quality analyzer (DeChem-Tech, Hamburg,
Germany). Temperature, pH and dissolved oxygen (DO) were measured
once in situ for each bucket every day, using a portable multiparameter
meter (520 M-01A, Thermo Orion, USA). Total organic carbon (TOC)/
total nitrogen (TN) of water in each bucket was analyzed every 10 days
using a total organic carbon analyzer (Multi N/C 2100, Analytikjena,
Germany).
S. Dong et al.
2.4. Analysis of microbial community dynamics
At the beginning of experiment settlement and every 10 days during
the rearing period, biofloc samples of experimental and control groups
were collected by filtration using 0.22 μm aqueous membranes sepa­
rately (Huang et al., 2020b). Also, at the end of 30 days, 10 shrimp from
each bucket were harvested and anesthetized according to the reported
method (Luedeman and Lightner, 1992). The intestines were aseptically
dissected on ice, and those from the same bucket were pooled to
compose a sample. Total genomic DNA from the bacterial community of
all biofloc and intestine samples was extracted using the HiPure Soil
DNA Kits (Magen, Guangzhou, China) according to manufacturer’s
protocol, and sent to Gene Denovo Biotechnology Co. (Guangzhou,
China) for sequencing of the 16 s rDNA V3-V4 region and community
structure analysis. (Primer sequence information 341F:
CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT); PCR
amplification products were cut and recovered, quantified using a
QuantiFluor™ fluorometer, and the purified amplification products
were mixed in equal amounts, sequencing junctions were ligated,
sequencing libraries were constructed, and Illumina PE250 was used for
sequencing analysis.
To obtain high quality clean reads, raw reads were further filtered
using FASTP (https://github.com/OpenGene/fastp) and paired end
clean reads were merged as raw tags by FLASH (version 1.2.11) with a
minimum overlap of 10 bp and mismatch error rates of 2% (Magoc and
Salzberg, 2011). Noisy sequences of raw tags were filtered to obtain
high-quality clean tags. 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). Then, filter tags whose continuous high-quality base length is
less than 75% of the tag length. After that the clean tags were clustered
into operational taxonomic units (OTUs) of ≥ 97% similarity using
UPARSE (version 9.2.64) pipeline. All chimeric tags were removed using
UCHIME algorithm (http://www.drive5.com/usearch/manual/uchime_
algo.html) and finally obtained effective tags for further analysis. The
tag sequence with the highest abundance was selected as a representa­
tive sequence within each cluster. Chao1, Ace, Shannon, Simpson,
Good’s coverage index were calculated in QIIME (version 1.9.1)
(Caporaso et al., 2010). Canonical correspondence analysis (CCA) was
executed in R project Vegan package (version 2.5.3).
2.5. Measurement of shrimp growth performance and enzyme activity
At the end of the experiment, the total number of survival shrimp in
each bucket were counted, and the mean final body weight was calcu­
lated. Thus, the survival rate (SR, %), specific growth rate (SGR, %) and
weight gain per week were calculated using the following equations:
SGR = (lnWt − lnW0 ) × 100/t
SR = Nt ⋅100/N0
( ) /
Growth rate mg⋅week− 1 = (Wt − W0 ) culture weeks
where Wt and W0 are the mean final and initial weight of shrimp
respectively, t is the duration of experiment time; Nt and N0 are the final
and initial number of shrimps, respectively.
At the end of 30 days, the hepatopancreases of 10 shrimp from each
bucket were collected for the physiological and biochemical analysis. All
shrimps were anesthetized according to the reported method (Luedeman
and Lightner, 1992), and then the hepatopancreas were excised with a
sterilized stainless-steel scalpel and tweezer. The excised hepatopan­
creas of P. vannamei were rinsed by ice-cold normal saline (0.8% NaCl),
and those from the same bucket were pooled to compose a sample, and
immediately frozen in liquid nitrogen and finally stored in a − 80 ◦ C
freezer. For future histological analysis, the frozen hepatopancreas were
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Aquaculture 554 (2022) 738057
quickly weighed in one 5 mL sterile tube and homogenized with the
same weight of ice-cold normal saline (0.8% NaCl) on an ice tray. The
homogenates were centrifuged at 4000 g for 15 min at 4 ◦ C, and the
supernatants were obtained and used for antioxidant parameters anal­
ysis. Glutathione peroxidase (GSH-Px), superoxide dismutase (SOD),
and catalase (CAT) activity, as well as malondialdehyde (MDA) content
were measured using relevant detection kits following the manufac­
turer’s protocols (Nanjing Jiancheng Institute of Biological Engineering,
China). GSH activity was measured by the reaction ability of dithodi­
nitrobenzoic acid with sulfhydryl compounds in producing a yellow
color, thus GSH-Px could be represented by catalyzed GSH reaction rate
by measuring absorbance at 412 nm for 5 min. The SOD activity was
assayed with the reaction based on its inhibition on the scale of super­
oxide anion generating by xanthine and xanthine oxidase reaction sys­
tem, and CAT activity was defined as the amount of enzyme that
decomposes H2O2. The MDA assay was evaluated based on the color
reaction between MDA and thiobarbituric acid. They were measured
using a microplate spectrophotometer (Synergy Neo2, Biotek, Wash­
ington, USA) at 412 nm, 550 nm, 240 nm, and 532 nm as described by
the manufacturer’s protocols, respectively.
2.6. Statistical analysis
All experiment data were analyzed in triplicate, and all graphs were
plotted by the Origin 2018 software (Origin Lab Corp., USA). All data
were analyzed for the significant difference (p < 0.05) by t-test per­
formed on SPSS Statistics software (version 19.0) and the standard de­
viation (STDEV) to reflect the dispersion degree of values relative to
mean.
3. Results and discussion
3.1. Water quality parameters
The water quality parameters of experimental and control groups
were shown in Table 1. There were no significant differences in pH, DO
and temperature between the two groups, all of which could fulfill the
requirements of shrimp growth. The mean of TOC/TN in the experi­
mental group was 4.52 ± 0.32, which was slightly higher than 4.07 ±
0.38 of the control group, although no significant difference was
observed. Currently, the C/N ratio for bioflocs cultivation are mainly
based on the previous results (Avnimelech, 1999; Ebeling et al., 2006)
and determined by exogenous additions of organic carbon, while the
reality is obviously more complex and water body can hardly maintain
high C/N ratios over a long period. Different strategies of organic carbon
addition based on the TAN content of water are also costly and un­
practical to maintain a high DOC/TAN in the long term (Luo et al.,
2019a). In contrast, microalgae, as primary producers, could serve to
increase the C/N ratio by sustainably synthesizing organic matter. In this
experiment, it seemed that the bioaugmentation of Platymonas sp. in­
crease the average TOC/TN of rearing water body, to a certain extent.
The contents of NO2-N and TAN in experimental and control groups
during the 30-day feeding were shown in Fig. 1A and Fig. 1B, respec­
tively. The TAN contents of two groups fluctuated in a similar trend, in
Table 1
Mean values (±SD) of water quality parameters in the microalgae experimental
group (MA) and control group (CK) during a 30-day rearing.
pH DO Temperature Salinity TOC/TN
(mg⋅L− 1) (◦ C) (g⋅L− 1)
7.72 ± 6.75 ± 4.52 ±
MA 28.4 ± 0.7 8.48 ± 0.22
0.17 0.46 0.32
CK
7.71 ± 6.81 ±
28.3 ± 0.7 8.28 ± 0.12
4.07 ±
0.16 0.32 0.38
Note: no significant difference was observed between MA and CK at p < 0.05
level.
S. Dong et al.
Fig. 1. Mean value (±SD) of (A) total ammonia nitrogen and (B) nitrite‑nitrogen in experimental group (MA) and control group (CK) during 30-day rearing.
which TAN accumulated gradually from the beginning, reached two
peaks on the 12th day and the 20th day, and then declined rapidly to the
24th day. The average TAN content of two groups had no significant
difference. The NO2-N contents of two groups maintained at an
extremely low level in the initial and middle stages, then reached a peak
from day 22 to day 24, and fluctuated within a relatively low concen­
tration. Overall, the average NO2-N contents in the experimental group
were significantly lower (p < 0.05) than that of the control group. This
result indicated that the bioaugmentation of Platymonas sp. significantly
reduced NO2-N accumulation in BFT system.
Many advantages of P. vannamei rearing in low salinity water have
been proposed, including minimizing the impact of diseases and short­
ening the distance to inland markets (Chang et al., 2015; Mariscal-
Lagarda et al., 2012). However, the lower salinity of the rearing envi­
ronment also decreases the tolerance of P. vannamei to inorganic nitro­
gen compounds (Valencia-Castaneda et al., 2019). Ammonia is the most
dominant and first pollutant produced by the direct excretion of aquatic
animals and the metabolic protein deamination of heterotrophic or­
ganisms for undigested food, feces and dead animals in rearing water
(Bartoli et al., 2008; Luo et al., 2020). The elimination of ammonia
mainly consists of assimilation by microbes to produce new biomass, as
well as nitrification metabolism including ammoxidation performed by
ammonia-oxidizing bacteria (AOB) and nitrite oxidation governed by
nitrite oxidizing bacteria (NOB) (Luo et al., 2020; Robles-Porchas et al.,
2020). For the role of microalgae in aquacultural water maintenance, it
was found that Platymonas helgolandica, Chlorella vulgaris and Chaeto­
ceros mulleri could significantly reduce TAN and NO2-N contents in a
zero-water exchange system, while the TAN and NO2-N reached to
excessive contents in the control group without a preculture of microbial
community (Ge et al., 2016). Pervious study validated that Nanno­
chloropsis oculata cocultivation with P. vannamei could improve rearing
water quality by controlling eutrophication and optimizing
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Aquaculture 554 (2022) 738057
microenvironment (Ding et al., 2021). With higher nitrogen uptake rates
and faster proliferation rates than AOB, microalgae were proved to be
superior competitors (Risgaard-Petersen et al., 2004). Recent study
showed that ammonium removal through assimilation of microalgae
was almost two times higher than that by nitrification (Bankston et al.,
2020). Meanwhile, in a microalgae-bacteria consortium, microalgae
supplied oxygen for the nitrification process, and bacteria excreted vi­
tamins or other active substances that enhanced the growth of micro­
algae (de Bashan et al., 2004; Ramanan et al., 2015). Following these
results, the experimental group had significant low NO2-N level
compared to the control group shown in this study, most probably
because of the enhancement of nitrogen assimilation and the promotion
of bacteria by Platymonas sp. bioaugmentation.
3.2. Analysis of the dynamic structure of the bacterial community
The species richness of the bacterial community was indicated by the
indexes of Sobs, Chao1 and Ace (Table 2). The bacterial species richness
in biofloc of the experimental group was gradually increased over time,
while the control group had a high species richness from 10 to 20 d. The
bacterial diversity of bioflocs in experimental and control groups both
reached a peak at 10 d as reflected by Simpson and Shannon indexes
(Table 2). The species richness and diversity of bioflocs and shrimp in­
testines in the experimental group was higher than those of the control
group at the same sampling time, and significant differences of alpha
diversity indexes were observed between experimental and control
groups (p < 0.05) on the 30th day. In summary, the bioaugmentation of
Platymonas sp. increased the species richness and diversity of bacterial
community in bioflocs. Similarly, previous studies demonstrated the
algae could increase the bacterial richness and diversity in the early and
late stage of biofloc-based P. vannamei culture systems (Jiang et al.,
2019), and the BFT system featuring green algae was more suitable for
S. Dong et al. Aquaculture 554 (2022) 738057

Table 2
Alpha diversity indexes of bioflocs and shrimp intestines samples from the experimental group (MA) and control group (CK) at different sampling times.
Time Sample Sobs Shannon Simpson Chao1 Ace Good’s coverage

MA1 1316.0 ± 161.1 5.55 ± 0.96 0.930 ± 0.052 1929.3 ± 205.8 1969.0 ± 193.7 0.995 ± 0.001
0d
CK1 1167.7 ± 27.2 4.98 ± 0.48 0.894 ± 0.032 1726.6 ± 22.6 1747.8 ± 53.3 0.996 ± 0.000
MA2 1468.7 ± 148.5 6.91 ± 0.23 0.977 ± 0.008 2198.0 ± 246.7 2182.8 ± 249.9 0.994 ± 0.000
10d
CK2 1446.3 ± 290.8 6.58 ± 1.27 0.959 ± 0.040 2169.9 ± 380.8 2168.6 ± 347.8 0.994 ± 0.001
MA3 1474.3 ± 117.2 6.38 ± 0.89 0.960 ± 0.027 2230.1 ± 223.7 2232.9 ± 179.2 0.994 ± 0.001
20d
CK3 1355.3 ± 157.5 5.42 ± 1.26 0.837 ± 0.178 2183.5 ± 274.3 2127.5 ± 272.5 0.994 ± 0.001
MA4 1523.7 ± 61.0* 6.48 ± 0.18* 0.966 ± 0.008 2355.8 ± 129.1* 2351.4 ± 104.6* 0.993 ± 0.000
CK4 1276.0 ± 67.0 6.05 ± 0.05 0.946 ± 0.014 1908.1 ± 132.4 1954.7 ± 117.3 0.995 ± 0.000
30d
MA-I 1080.3 ± 25.6* 6.15 ± 0.08 0.960 ± 0.006 1685.0 ± 167.8 1719.9 ± 159.6 0.997 ± 0.000
CK-I 923.7 ± 88.5 5.71 ± 0.55 0.954 ± 0.015 1342.9 ± 152.6 1392.0 ± 165.3 0.997 ± 0.001

Note: the asterisk (*) represented significant difference between MA and CK at p < 0.05 level. MA1–4 and CK1–4 are biofloc samples on day 0, 10, 20 and 30, and MA-I
and CK-I are shrimp intestines samples, respectively.

shrimp growth and more conducive to manipulating a favorable bac­
terioplankton community (Yang et al., 2020).
The bacterial 16S rRNA gene of each group at different times were
sequenced to investigate the structure and succession procedure of mi­
crobial communities. In all samples, the dominant phyla (>1% in any
sample) of bacterial communities were Proteobacteria, Bacteroidetes,
Actinobacteria, Planctomycetes, Verrucomicrobia, Patescibacteria, and
Cyanobacteria, and the total relative abundance of these dominant
phyla exceeded 95% in each sample (Fig. 2). A total of 553 genera were
detected across the samples, and the heat map of 35 dominant genera (>
1% in any sample) was generated in Fig. 3. In the early stage, Pseu­
dooceanicola, Phaeodactylibacter, Candidatus Aquiluna were mainly pre­
sent in the experimental group, Tistlia were mainly present in the control
group, while Enterobacter and Aestuariibacter were dominant in both two
groups. Subsequently, Rheinheimera, Marinibacterium, Denitromonas,
Prosthecobacter, Mycobacterium, Nannocystisand and SM1A02 were
gradually abundant in the experimental group and became the dominant
microorganisms (10–20 d). Towards the end of rearing time, the abun­
dance of Tamlana Cellvibrio and Fulvivirga was observably increased in
the experimental group, while Marivita, Algoriphagus, Erythrobacter Sal­
inarimonas, and Vibrio were dominated in the control group. Meanwhile,
Cellvibrio and Fulvivirga became the dominant microorganisms in both
bioflocs and shrimp intestines in the experimental group at the later
stages. In contrast, Vibrio, most of which were regarded as opportunistic
pathogens for shrimp, were dominant in bioflocs and shrimp intestine of
the control group, suggesting an inhibition of Vibrio by microalgae
bioaugmentation. This was consistent with previous studies that the
Vibrio concentration in rearing water, sediment, stomach, and intestines
was significantly reduced by adding microalgae under different culture
systems (Ge et al., 2016; Huang et al., 2022).
Fig. 2. Composition and abundance of major bacterial groups at the phylum
level (>1% in any sample) in bioflocs of the experimental group (MA) and
control group (CK) on day 0, 10, 20 and 30 (MA1–4 and CK1–4, respectively),
and shrimp intestines of MA and CK on day 30 (MA-I and CK-I).
The microbiological functional change of bioflocs was associated
with the bacterial community difference between experimental and
control groups after the Platymonas sp. bioaugmentation. Among the
significantly increased microbial species in the experimental group
compared with the control group, Prosthecobacter is known as a nitro­
sification bacteria while SM1A02 is classified as nitrifying bacteria (Gu
et al., 2020; Luo et al., 2019b; Schveitzer et al., 2020). Denitromonas,
Rheinheimera and Mycobacterium advantaged in A2 and A3 samples were
reported to play important roles in denitrifying process (Commault
et al., 2017; Meng et al., 2020; Yao et al., 2021; Zhang et al., 2019).
Thus, the bioaugmentation of microalgae potentially enhanced nitrifi­
cation and denitrification in BFT system. Phaeodactylibacter and Rose­
obacter dominated in A1 and AG samples were reported to have
antagonistic activity against Vibrio anguillarum (Grotkjær et al., 2016; Li
et al., 2020; Porsby et al., 2008). Besides, the stability of Platymonas sp.
bioaugmentation possibly benefit from the increased abundance of these
bacteria closely related to microalgae supplement. SM1A02 and Cellvi­
brio, which were enriched in the experimental group, was applied to
promote the production of microalgal biomass and specific metabolites
with a co-cultivation method (Liu et al., 2020; Xie et al., 2021). More­
over, several flocculating bacteria including Cellvibrio and Nannocystis
enriched in the experimental group could be helpful to the formation
and functional maturity of bioflocs in shrimp culture system (Wang
et al., 2020; Zhang et al., 2002).
To clear the relationship between environmental factor dynamics
and bacterial community variations, CCA (canonical correspondence
analysis) was performed with top 20 dominants at the genus level
(Fig. 4). Since the beginning, samples from two groups moved from the
third quadrant to the lower right, then gradually to the upper right with
the experimental duration. The combination of environmental factors
and dominant species analysis showed that the early-stage samples were
positively correlated with temperature, dissolved oxygen, and TAN
other than NO2-N, which also had strong positive correlations with
Prosthecobacter, Marinibacterium, SM1A02, and Denitromonas mainly
dominated in A2 of experimental group compared to control group
(Fig. 4). Uniformly, Prosthecobacter, SM1A02, and Denitromonas, were
closely associated to nitrification and denitrification as reported in the
previous study (Gu et al., 2020; Luo et al., 2019b; Meng et al., 2020;
Schveitzer et al., 2020). The dominance of these bacteria could accel­
erate the utilization of ammonia nitrogen in the experimental group
with Platymonas sp. bioaugmentation. In contrast, the negative corre­
lation between bacterial and NO2-N content was observed in this study,
meanwhile the dominant microorganisms positively associated with
NO2-N n included Shewanella, Erythrobacter, and Hoppeia, which were
largely dominant genera in C4 of the control group compared to the
experimental group with Platymonas sp. bioaugmentation (Fig. 4).
Erythrobacter species were frequently isolated from nutrient-rich envi­
ronment and involved in carbon cycling and carotenoids production
(Asker et al., 2018). Species of the genus Shewanella were reported to
have antagonistic abilities against Vibrio species and modulate

5
S. Dong et al. Aquaculture 554 (2022) 738057

Fig. 3. Heatmap plot of the dominant genera (> 1% in any sample) in bioflocs of the experimental group (MA) and control group (CK) on day 0, 10, 20 and 30
(MA1–4 and CK1–4, respectively), and shrimp intestines of MA and CK on day 30 (MA-I and CK-I).

immunological genes of aquacultural animals (Interaminense et al.,
2019; Wei et al., 2021). In the later stage of the experiment, the insuf­
ficient or inhibited nitrite oxidation gradually led to the NO2-N accu­
mulation and the increased water nutrient, which might cause the
emergence of Vibrio in the control group (Fig. 4), and then the abun­
dance of Vibrio-antagonism probiotics (such as Shewanella) rose
correspondingly.
3.3. Growth performance and antioxidant capacity of shrimps
The growth performance of shrimp during the experimental period
was shown in Table 3. Significant difference was observed (p < 0.05) in
survival rate between the experimental group and the control group. On
the 30th day, the mean body weight of shrimp in the experimental group
(726.8 ± 226.8 mg) was 152% heavier than that of the control group
(475.8 ± 248.2 mg) with a significant difference (p < 0.05), as well as
the SGR and weekly weight gain. The antioxidant enzyme activities and
MDA contents of shrimp hepatopancreas in this study were shown in
Fig. 5. In the experimental group, enzyme activities of SOD, CAT and
GSH-Px were significantly higher than those in the control group,
respectively (p < 0.05), while no significant differences were observed
between two groups in MDA content. Overall, these results suggest the
bioaugmentation of microalgae were beneficial to the zootechnical
performance and antioxidant response of shrimps.
Numerous researches have demonstrated that microalgae in a cul­
ture system or as aquafeed ingredients have great benefits for aquacul­
tural animals. The addition of Platymonas helgolandica enhanced the
growth performance of P. vannamei, as well as total hemocyte count
(THC), phenoloxidase activity (PO), lysozyme (LSZ), and SOD activity in
a zero-water exchange system (Ge et al., 2017). Recent study also re­
ported the growth performance, antioxidant capacity, and immunity of
Artemia nauplii were enhanced by feeding a diverse group of live
carotenoid-rich microalgae (Gui et al., 2022). The addition of two fila­
mentous microalgae as feed ingredients for Carassius auratus gibelio had
significant effects on tissue fatty acid profiles, antioxidant capacity and
immunity with the same growth performance (Chen et al., 2019). In the
cultivation of shrimp, cellular metabolism and phagocytic process
eventually results in the production of ROS, including hydrogen
peroxide (H2O2), superoxide anion (O2− ), and hydroxyl radical (⋅OH)
(Kuebutornye et al., 2019). Excessive amounts of ROS can damage
macromolecules (e.g., proteins, lipids, and DNA) and cell membranes of
shrimp (Zeinali et al., 2015). As a major part of the antioxidative defense
system, enzymatic components (including SOD, CAT, GSH et al.) catal­
yse the dismutation of ROS (Wang et al., 2011). The antioxidant defense
system and immune system of shrimp are closely linked (Xu and Pan,
2013). An enhanced antioxidant status could facilitate shrimp immune
defense function and finally promote the zootechnical performance of
shrimp (Castex et al., 2010; Chiu et al., 2007). Whereupon, the current

6
S. Dong et al. Aquaculture 554 (2022) 738057

Fig. 4. Canonical correspondence analysis with water parameter, biofloc communities and top 20 dominants. TAN, total ammonia nitrogen; NO2-N, nitrite‑nitrogen;
Temp, temperature; DO, dissolved oxygen; C/N, total organic carbon/total nitrogen; Sal, salinity. Arrows represented water parameters as response variables; circles
represented dominant bacteria as factor explanatory variables; triangles represented biofloc communities as factor explanatory variables.

Table 3
Mean values (±SD) of the zootechnical performance of Penaeus vannamei in the
experimental group (MA) and control group (CK) on day 30.
SR (%) Initial Final SGR (%) Growth rate
weight (mg) weight (mg) (mg⋅week− 1)

MA 90.3 ± 2.3 ± 0.1 726.8 ± 19.11 ± 169.04 ± 5.20*

1.5* 226.8* 0.10*
CK 55.3 ± 2.3 ± 0.1 475.8 ± 17.75 ± 110.48 ± 14.71
3.2 248.2 0.45

Note: the asterisk (*) represented significant difference between MA and CK at p

< 0.05 level.

results indicated that the bioaugmentation of Platymonas sp. was

conducive to the improvement of growth performance and antioxidant
capacity of P. vannamei reared in BFT system.
The enhancement of the BFT system by the addition of microalgae
can not only improve the zootechnical performance of shrimp, but also
serve the economic interests of the shrimp aquaculture industry
(Nagappan et al., 2021). To elaborate, microalgae has been utilized as a Fig. 5. Relative antioxidant enzyme activities (SOD, CAT and GSH-Px) and
sustainable feed ingredient in aquaculture for years, providing high MDA contents (±SD) of shrimp hepatopancreas in the experimental group (MA)
nutritional protein, beneficial pigments (such as chlorophyll, lutein, and control group (CK) on day 30. The asterisk (*) represented significant
difference between MA and CK at p < 0.05 level.

7
S. Dong et al.
astaxanthin, and β-carotene), polyunsaturated fatty acids, vitamins, and
minerals (Nagarajan et al., 2021; Rajesh Banu and Preethi, 2020). The
continuous addition of microalgae could reduce the amount of feedstuff
used and improve the nutritional quality of shrimp (Huang et al., 2022;
Nagarajan et al., 2021), thus contributing to the attainment of higher
economic benefits. Furthermore, the enhancement method proposed in
this study bypasses the solid-liquid separation after microalgae culti­
vation, which is currently regarded as a major economic bottleneck in
microalgae meal production (Nie et al., 2020). So that even though
additional equipment purchases and labor costs have to be incurred, the
local microalgae cultivation is still a more cost-effective addition
method than microalgae meal supplementation. Also, by enhancing the
BFT system with cultured microalgae, the operation cost will be reduced
by maintaining a higher water quality with lower exogenous organic
carbon added (Dong et al., 2021). Ultimately, it’s necessary to investi­
gate the viability and economics of microalgae enhancement in a full-
scaled BFT system in future studies.
4. Conclusions
The results of this study indicate that the bioaugmentation of Platy­
monas sp. with a concentration of 105 cell⋅mL− 1 is beneficial for water
quality management by affecting the microbial community, and anti­
oxidant capacity and nursery performance of P. vannamei in BFT system.
Current findings preliminarily proved the application value of micro­
algae in BFT and provided a new direction for the set-up and mainte­
nance of microbial ecological function in BFT systems.
Credit authorship contribution statement
Sheng Dong: Conceptualization, Data curation, Formal analysis,
Methodology, Project administration, Validation, Visualization, Writing
- original draft, Writing - review & editing. Yu Li: Data curation, Formal
analysis, Validation. Fei Huang: Formal analysis, Writing - original
draft, Writing - review & editing. Langli Lin: Data curation, Method­
ology, Validation, Visualization. Ze Li: Validation, Visualization. Jian­
cheng Li: Data curation, Formal analysis. Yuxiu Zhang: Supervision,
Project administration, Funding acquisition. Yihong Zheng: Concep­
tualization, Formal analysis, Investigation, Methodology, Supervision,
Validation, Visualization, Writing - original draft, Writing - review &
editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work was financially supported by the Guangdong Natural
Science Foundation (2020A1515010873), Shenzhen Special Program
for Development of Emerging Strategic Industries
(JSGG20180130103422600), the National Natural Science Foundation
of China (31870343), Innovation-Driven Development Special Fund
Project of Guangxi (Guike AA18242047), Grant Plan for Demonstration
City Project for Marine Economic Development in Shenzhen (No. 86) to
Dr. Zhangli HU, and the Fundamental Research Funds for the Central
Universities of China University of Mining and Technology (Beijing)
(Grant No. 2021YJSHH30).
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