Aquaculture and Fisheries 3 (2018) 246–253

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Aquaculture and Fisheries

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a q u a c u l t u r e - a n d - fi s h e r i e s /

Performance of feeding Artemia with bioflocs derived from two types of fish
solid waste
Miaolan Yao a, Guozhi Luo a, b, c, *, 1, Hongxin Tan a, b, c, Lipeng Fan a, Haoyan Meng a
a
Research and Development Center of Aquacultural Engineering of Shanghai, Shanghai, 201306, China
b
Shanghai Collaborative Innovation Center for Aquatic Animal Genetics and Breeding, Shanghai, 201306, China
c
National Demonstration Center for Experimental Fisheries Science Education (Shanghai Ocean University), Shanghai, 201306, China

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

Keywords: The production of bioflocs with the solid waste from recirculating aquaculture systems (RAS) and feeding Artemia
Bioflocs results in additional nutrient retention and lowers waste discharged from RAS. The solid waste from the drum-
Fish waste filters of two RAS, which stocked European eel (Anguilla anguilla) and Nile tilapia (Oreochromis niloticus), was
Artemia
used as substrate to produce bioflocs in suspended growth reactors, referred to as E-flocs and T-flocs, respectively.
Recirculating aquaculture system
Suspended growth reactors
Mono-diets consisting of 100% E-flocs and 100% T-flocs were added to culture Artemia, referred as E-Artemia and
T-Artemia, respectively, in a laboratory scale test. The efficiency of this feeding regime was investigated. A sig-
nificant difference was observed in terms of crude protein content (35.59
0.2%) for E-flocs, (29.29
0.95)% for
T-flocs, (70.01
0.92)% for E-Artemia and (65.63
0.89)% for T-Artemia. 134 out of the total operational
taxonomic units (OTUs) were present in E-flocs and T-flocs from the analysis of high-throughput sequencing data.
Most of the shared OTUs belonged to cyanobacteria. C18:1n7 of T-flocs was higher than that of E-flocs (P < 0.05).
C18:2n6 of E-flocs was significantly higher than that of T-flocs (P < 0.05). No significant difference was observed
in the other fatty acid compositions (P > 0.05). The survival rate of E-Artemia was (22
0.02) %, significantly
higher than that of T-Artemia (16%
0.02%) (P < 0.05). No significant difference was observed between the
average body weight of E-Artemia (2.38
0.40 mg) and E-Artemia (2.91
0.21) (P > 0.05). The EPA of Artemia
fed with E-flocs was (3.00
0.46)%, significantly higher than that of T-Artemia (1.57
0.19%) (P < 0.05). This
study offers a method for reusing the aquaculture waste, which will be helpful to achieve a zero-pollution
discharge for aquaculture systems.

1. Introduction applied feed was discharged as waste sludge (Davidson & Summerfelt,
2005). As the result of the super-intensive culture in RAS, a considerable
Aquaculture is one of the fastest growing animal food-producing amount of sludge is produced that must be treated before it can be
sectors (FAO, 2014). To obtain higher yields, several intensified aqua- disposed (Mirzoyan, Tal, & Gross, 2010; Timmons & Ebeling, 2007).
culture systems were developed through a high stocking density and Waste characteristics may vary widely, depending on the fish species
inputs of artificial food with 25%–45% crude protein (Kutty, 2005). (Timmons & Ebeling, 2007). European eel (Anguilla Anguilla) and Nile
Shrimp or fish naturally utilize a limited amount of the nutrition con- tilapia (Oreochromis niloticus) are two of the common species farmed in
tained within the food, with up to 75% of the input nitrogen discharged RAS (Martins et al., 2010; Mota, Martins, Eding, Can
ario, & Verreth,
(Hargreaves, 1998; Tovar, Moreno, Manuel-Vez, & GarciaVargas, 2000). 2017).
If the nitrogen is not treated properly before being discharged, it can be Biofloc technology (BFT) uses aerobic heterotrophic bacteria to
detrimental to the environment. In response to this concern, recirculating convert inorganic nitrogen (NHþ  
4 -N, NO2 -N and NO3 -N) into micro-
aquaculture systems (RAS) were explored as an alternative to pond and biomass, which is a protein source for the fish or shrimp (De Schryver
cage culture systems (Timmons & Ebeling, 2007). Although RAS provide & Verstraete, 2009). Biofloc is a heterogeneous aggregate of bacteria,
many advantages over traditional aquaculture systems, 11%–40% of the bacteria-grazing protozoans, exocellular polymers, and particulate

* Corresponding author. Research and Development Center of Aquacultural Engineering of Shanghai, Shanghai, 201306, China.
E-mail address: gzhluo@shou.edu.cn (G. Luo).
1
Current position and address: Hucheng Ring Road 999, Shanghai Ocean University, Shanghai 201306, China.

https://doi.org/10.1016/j.aaf.2018.07.002
Received 14 January 2018; Received in revised form 7 July 2018; Accepted 10 July 2018
Available online 1 August 2018
2468-550X/© 2018 Published by Elsevier B.V. on behalf of Shanghai Ocean University. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
M. Yao et al. Aquaculture and Fisheries 3 (2018) 246–253

organic matter, which is often included in the diets either directly for Artemia (Fig. 1).
filter feeding or omnivorous species, such as shrimp and tilapia (Har-
greaves, 2013; Xu, Morris, & Samocha, 2016), or in a processed feed for
shrimp (Kuhn, Lawrence, Crockett, & Taylor, 2016). However, the pro- 2.2. RAS, fish waste and SGBRs
cessing always requires drying, milling, and storage, which can affect
nutritional characteristics (Emerenciano, Gaxiola, & Cuzon, 2013). The initial solid fish waste was obtained from the solid/liquid sepa-
Artemia is one of the best sources of live food for cultured fish and rators of two RASs stocked with European eel (Anguilla Anguilla) and Nile
shellfish species (Toi, Boeckx, Sorgeloos, Bossier, & Stappen, 2013). tilapia (Oreochromis niloticus) in Recirculating Aquaculture Engineering
Artemia can feed on a wide range of diets, such as micro-algae, bacteria, and Technology Laboratory, Shanghai, China. The RASs were equipped
protozoa, and small detritus particles (Fern
andez, 2001). Artemia has with eight 1 m3 tanks, a drumfilter separator, a cold/heat control, a gas/
been cultured using chicken manure, pig manure, and algae-rich green liquid mixing device, two biofilters, an ultraviolet sterilization unit, and
water as food sources (Sui et al., 2013; Verma, Raghukumar, & Naik, two circulating pumps (Fig. 1). The water in the system was recirculated
2011). Previous research has proved that Artemia can efficiently feed on at a rate of 24 times per day. Root-type compressed air blowers were used
the bioflocs produced from aquaculture waste (Luo, Yao, Tan, & Wu, to aerate the water. The stocking density of eel and tilapia in each RAS
2017). This makes it possible to avoid the risks of adverse environmental was approximately (12
0.5) kg/m3 and (22.6
1.0) kg/m3,
impacts from aquaculture processing. Moreover, feeding Artemia with respectively.
bioflocs may be a promising way to directly deliver probiotics to the The eels were fed a commercial pellet diet (Tongwei 8912, Sichuan,
digestive tract of the target aquaculture species (Kesarcodi-Watson, China) containing 10% moisture, 48% crude protein, 2% crude fibers,
Kaspar, Lategan, & Gibson, 2008; Suzer et al., 2008). 17.0% crude ash, and 1.5% total phosphorus. The tilapias were fed a
Suspended-growth batching reactors (SGBRs) were connected to the commercial pellet diet (Tongwei 8912, Sichuan, China) containing 10%
drum-filter of the RAS to produce bioflocs with solids waste (Azim, Little, moisture, 30% crude protein, 12% crude fibers, 13% crude ash, and 1.5%
& Bron, 2007; Luo et al., 2017; Schneider, Sereti, Machiels, Eding, & total phosphorus.
Verreth, 2006). The potential of this technology stems from the addi- The characteristics of eel waste were as follows: (147.6
2.2) g/L of
tional protein retention harvested and lower overall nutrient discharge total suspended solids (TSS), (238.8
11.3) g/L of chemical oxygen
from RAS (Schneider et al., 2006). The current experiment was designed demand (COD), and (9.9
0.3) g/L of nitrogen. The characteristics of
to investigate (1) bioflocs production using two types of RAS solid waste tilapia waste were as follows: (138.2
1.2) g/L of TSS, (308.6
10.1) g/
and (2) the efficiency of the above two types of bioflocs to feed Artemia. L of COD, and 5.1 g/L of nitrogen. The nitrogen content of the bioflocs
was used instead of the crude protein content because all the organic
2. Material and methods nitrogen was degraded to ammonium nitrogen first and then assimilated
into the bioflocs as micro-biomass (Avnimelech, 1999).
2.1. Overview of experiment strategy Six SGBRs, with a working volume of 15 L, were used to produce
bioflocs in the current study, three for tilapia waste (T-flocs) and three for
A laboratory-scale integrated RAS-SGBRs-Artemia production system eel waste (E-flocs). The fish waste from the drum-filters of RASs were
was established for the current experiment. The system was comprised of completely pulverized and filtered through a 10-μm mesh and the filtrate
three compartments: (a) two RASs culturing Nile tilapia/European eel; was added to each reactor in equal amounts as the TSS source at initial
(b) six SGBRs producing bioflocs; and (c) six cylinders rearing Artemia. concentrations of approximately 5000 mg/L. Each reactor was filled with
Water and sludge collected in the solid/liquid filter was batch-fed into seawater with salinity (30
1.0) g/L. The temperature of the reactors
the SGBRs periodically to produce bioflocs, which were used as food for was maintained at (25
1)  C. The mixture was thoroughly suspended
by placing an air stone at the bottom of each reactor. The air blowers

Fig. 1. The technical process diagram of the current experiment.

247
M. Yao et al. Aquaculture and Fisheries 3 (2018) 246–253

were supplied with a 138-W air pump (ACO-008, SenSen Co., Ltd., Survival (%) ¼ (final number of Artemia/initial number of Artemia)  100.
Zhejiang, China) operating at a rate of approximately 20 L/min. Dis-
solved oxygen (DO) was maintained (7.2
1.0) mg/L. The dissolved The biomass production (BP) in wet weight (g/L) in each cylinder was
organic carbon to total ammonia nitrogen ratio (DOC/TAN) in the calculated by weighing the total production and the averaged BP for
SGBRs-BFT was maintained at >15 (w/w) by adding glucose as the every diet.
external carbohydrates.
The SGBRs reached a steady state on approximately day 37 (Har- 2.4.3. Crude protein and fat content of Artemia and bioflocs
greaves, 2013). From day 38, the bioflocs were harvested from the SGBRs Artemia and microbial flocs used to determine crude protein content
to feed Artemia, and the same weight of fresh waste from the RAS was and crude fat content were freeze-dried at 80  C before analysis. The
added every day. The crude protein content, and the quantity and quality crude protein content was determined by measuring nitrogen via the
of the fatty acid (FA) of flocs were measured at every feeding time. Kjeldahl method (Chinese SEPA, 2004). The protein content was calcu-
Glucose was added to the SGBRs every day to enrich the flocs to maintain lated assuming that it contained 16% nitrogen and was expressed as a
DOC/TAN at > 15 (w/w) during the feeding period. percentage of the bioflocs’ dry matter weight (AOAC, 1999). The volatile
suspended solids (VSS) fraction was considered as a measure of the
2.3. Artemia bacteria concentration in the current experiment (Tchobanoglous, Bur-
ton, & Stensel, 2003).
The enrichment grade Artemia cysts (Shanghai Ocean University,
China) were hydrated in tap water for 1 h at 4  C using a standard pro- 2.4.4. Fatty acid extraction and analysis
tocol, and then the cyst shells were decapsulated (Sorgeloos, Lavens, Crude fat was extracted from the sampled Artemia as described by
eger, Tackaert, & Versichele, 1986). The decapsulated cysts were
L
Folch, Lees, and Sloane Stanley (1957). The samples, coupled with 50 mL
incubated at a density of 30 g/L in a 160-L conical glass at (27
1)  C, pH of nonadecanoic acid, were used as substitute then added with standard
(8.50
0.50), salinity (35
1) g/L, and continuous light of 1000 lx for working solution. The samples were lapped by pellet pestle (Sigma-Al-
24 h. drich, USA), homogenized with chloroform/methanol (2/1) to a final 20
The Artemia instar I nauplii were separated from the empty cysts, times volume and stored at 4  C overnight for fat extraction. After
washed in tap water, and placed in 4-L plastic cylinders containing well- centrifugation at 2000 r/min for 10 min, the supernatant was transferred
aerated seawater (salinity: 30
1) at (26
1)  C. The cylinders were to 50 mL storage bottles and thoroughly homogenized with 3 mL of
aerated to supply dissolved oxygen (up to 6 mg/L). All of the cylinders saturated saline using the pellet pestle (Sigma-Aldrich, USA) on ice for at
were irradiated with an illumination of 1000 lx on a 12 h/12 h on/off least 2 h. he upper liquid phase layer was then removed and the lower
cycle. A stocking density of 3000 nauplii/L was employed in each tank. layer organic phase layer mixed with 1 mL of the chloroform: methanol:
The two dietary treatments were set up in triplicate. Dietary E-flocs water (v: v: v ¼ 3:48:47) solution. After discarding the upper layer, the
contained only bioflocs produced from eel waste. Dietary T-flocs con- extracts were evaporated under a moderate nitrogen flow until dry. The
tained only bioflocs produced from tilapia waste. Crab, Lambert, residuum was mixed with 2 mL of boron trifluoride methanol solution
Defoirdt, Bossier, and Verstraete (2010) suggested that the bioflocs at the (14%, w/w) in every test tube followed by water bath at 100  C for 20
concentrations of 0.18 g/L could be eaten efficiently by nauplii. The min, before immediate transfer into cold water. After that, 2 mL of
bioflocs were harvested directly from the SGBRs and fed to Artemia in the benzene and 2 mL of methanol saturated normal saline were added into
doses of 0.2–0.5 g/L suspended solids, at intervals determined by the each tube and mixed with a violent shake in a water bath at 100  C for 20
observation of Artemia intestines. Artemia was cultured over a period of min, and then the supernatant was transferred into 50 mL stripping tubes
19 days and checked daily for the ingestion status under a binocular and eluted using 2 mL n-hexane followed by an addition 4 mL of
microscope. Uneaten flocs and wastes from Artemia were not removed n-hexane. The last procedure was repeated once. The resulting extracts
and no water was changed during the experimental period. The indi- were evaporated until dry under a nitrogen stream and transferred into 2
vidual length, survival rate, biomass production, fatty acid (FA) content, mL glass tube. Each stripping tube was washed with n-hexane three
and crude protein content of Artemia in each dietary trial were analyzed times, with the washing liquid transferred into glass tubes. All extracts
at the end of the experiment. were evaporated until dry under a nitrogen stream to 1 mL in a water
bath at 40  C, transferred into sample vials, sealed with parafilm, and
stored in darkness at 20  C until the GC-MS analysis.
2.4. Data collection and sample analysis
A 7890A gas chromatograph coupled with a 5975C mass spectrom-
eter (Agilent Inc., USA) was used. The FA methyl ester was separated
2.4.1. Water quality parameters
using a DB-5 ms capillary column (30 m  0.25 mm  0.25 μm, Agilent
The water temperature ( C), DO, and pH were measured daily using a
Inc., USA). The identification and quantification of the fatty acids were
YSI-pro plus meter (YSI Incorporated, Yellow Springs, OH, USA). The
performed according to the method of Toi et al. (2013). The FAs were
TSS, volatile suspended solids (VSS), NHþ  
4 -N, NO2 -N and NO3 -N were
identified by comparing the peak area of each peak with the peaks of the
analyzed according to the Chinese State Environmental Protection
known standards (Guinot et al., 2013), 0.2 g fresh tissue þ1.0 mL
Agency (SEPA) standard methods (Chinese SEPA, 2004). The dissolved
(1NH2SO4) þ 100 μg internal standard (19:0 methylester,10 μL from 10
organic carbon (DOC) was evaluated daily using a total organic carbon
mg/mL) in a screw-capped tube.
analyzer (TOC-V CPH; Shimadzu Seisakusho, Japan).

2.4.5. Bacteria community analysis

2.4.2. Artemia performance parameters
On day 38, the bioflocs were taken out of SGBRs for the bacterial
Ten Artemia from each dietary trial were randomly collected. The
community analysis. In addition, Artemia adults were kept in ethanol
individual lengths of the Artemia, from the front of the head to the end of
(70%) and washed three times with sterile ultrapure water at the end of
the telson, were determined using an electronic dissecting microscope
the experiment. They were dissected with sterile tools under a stereo-
(Nikon SMZ1500, Nikon Instruments Inc., Melville, NY, USA) and con-
scope microscope to isolate the guts, which were homogenized with a
verted to the actual length using software Artemia 1.0® (courtesy of
pipette by blowing up and down until complete homogenization of the
Marnix Van Domme). After being rinsed thoroughly in deionized water to
soft Artemia tissues was achieved. Then, the Artemia adults were incu-
remove waste, the Artemia were then placed on tissue paper to remove
bated with lysozyme (Sigma) for 45 min. The genomic DNA was
excess water and then weighed. All Artemia were harvested at the end of
extracted using the Qubit 2.0 DNA and the polymerase chain reaction
the experiment. The survival in each replicate was calculated according
products were determined by pyrosequencing using Miseq Illumina by
to the following equation:

248
M. Yao et al. Aquaculture and Fisheries 3 (2018) 246–253

Majorbiotech Co., Ltd. (Shanghai, China) according to the standard Following sub-sampling, a total of 39853 sequences with an average
protocols and software (data collection software, Illumina). The raw length of 430 bp for E-flocs and a total of 30239 sequences with an
sequencing data obtained from this study were deposited to the NCBI average length of 433 bp for T-flocs were acquired. These results indi-
Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/) with the cated that there were no significant differences in the species diversity
project accession number of SRR5366148 - SRR5366151. (Shannon index), the species richness indices of ACE and Chao1 between
The sequences were clustered into OTUs by setting a 0.03 distance E-flocs and T-flocs (Table 2). The total classified OTUs in the bacterial
limit (equivalent to 97% similarity) using the UCLUST v1.1.579. The communities were 162 and 179 for E-flocs and T-flocs, respectively. 134
Shannon index, Chao 1 index, and abundance-based coverage estimator out of the total OTUs were present in E-flocs and T-flocs. Most of the
(ACE) indices were calculated to compare the microbial diversity and shared OTUs belonged to cyanobacteria.
richness between these flocs samples. Sequences were phylogenetically At the phyla level, the dominant bacteria communities were similar,
assigned to taxonomic classifications using an RDP classifier Bayesian and the abundance showed a little difference. The top three phylum in
Algorithm (http://rdp.cme.msu.edu/). The sequences were allocated terms of average abundance of E-flocs were cyanobacteria (60.62%),
down to the phylum, class, and genus levels. The relative abundance of a Proteobacteria (20.00%), and Bacteroidetes (9.48%). The top three
given phylogenetic group was set as the number of sequences affiliated phylum in terms of average abundance of T-flocs were cyanobacteria
with that group divided by the total number of sequences per sample. (49.79%), Proteobacteria (26.58%), and Bacteroidetes (11.32%).
There were 11 out of 135 genera with relative abundances higher
2.5. Data analysis than 1% for E-flocs. The top three genera, in terms of average relative
abundance of E-flocs, were Parvibaculum (60.61%), Propionibacter-
Statistical analysis of the experimental data was conducted using iaceae (6.69%), and Bradyrhizobium (3.93%). There were 13 out of 128
SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). The data of crude genera with relative abundances higher than 1% for T-flocs. The top
protein content, fatty acid composition of bioflocs and Artemia, survival three genera, in terms of average abundance of T-flocs, were Parvibac-
rates of Artemia were checked for homogeneity of variance and normal ulum (49.79%), Dietzia (8.42%), and Bradyrhizobium (6.14%). Parvi-
distribution by Levene’s F test and P-P plot. The data failed to meet these baculum was the most dominant genera in both E-flocs and T-flocs (Fig.
assumptions and were logarithmically transformed to satisfy normal 2).
distribution and to homogenize variance. An ANOVA followed by Tukey
post hoc test at 0.05 probability level was employed for the parameters of 3.3. Artemia performance
two types of flocs and each feed regime.
The survival rate (22
0.02%) of Artemia fed E-flocs (E-Artemia) was
3. Results significantly higher than that (16
0.02%) of fed T-flocs (T-Artemia) (P
< 0.05). No significant differences in individual length, wet weight, and
3.1. Nutritional quality of biofloc biomass produced were observed between the two groups (P > 0.05)
(Table 3).
There were no observed differences in the VSS concentrations of E-
flocs and T-flocs (P > 0.05) (Table 1). The crude protein content of T-flocs 3.4. Crude protein content and fatty acid composition of Artemia
(35.59
0.23%) was significantly higher than that of E-flocs (29.29%

0.95%) (P < 0.05). The biofloc was further analyzed to see whether it There was a significant difference in the crude protein content be-
contained essential fatty acids on day 37 (Table 1). There were no sig- tween E-Artemia and T-Artemia (P < 0.05), (70.01
0.92)% and (65.63

nificant differences among the other compositions between E-flocs and T- 0.89)%, respectively (P < 0.05) (Table 4).
flocs (P > 0.05), except for the C18:1n7 and C18:2n6. At the end of the culture period, the fatty acid analysis of Artemia
suggested that C18:1n9c of T-Artemia was significantly higher than that
3.2. Diversity of bacteria community in bioflocs of E-Artemia (P < 0.05). The other fatty acids of E-Artemia were slightly or
significantly higher than those of T-Artemia (P < 0.05). It is worthy to
The Illumina MiSeq sequencing was applied to evaluate the differ- highlight that C20:5n3 (Eicosapntemacnioc Acid, EPA) was (3.00
0.46)
ences in the bacteria community composition of E-flocs and T-flocs. A % in E-Artemia, which was higher than that of T-Artemia ((1.57

97% (cut off ¼ 0.3) was used to cluster OTUs in the downstream analyses. 0.19)%) (P < 0.05).

Table 1 3.5. Bacteria community in the gut of Artemia

Concentrations of total suspended solids (TSS), volatile suspended solids (VSS)
and crude protein content, Fatty acid composition (>1%) of T-flocs and E-flocs. The top three in terms of phyla of the bacteria community in the gut of
E-flocs (%) T-flocs (%) E-Artemia were cyanobacteria (43.21%), Proteobacteria (43.45%), and
TSS (mg/L) 5149.33
341.90a 5266.00
373.77a
Firmicutes (16.13%). 10 out of 175 genera had relative abundances
VSS (mg/L) 1492.00
34.95a 1591.83
57.36a higher than 1% for the gut of E-Artemia. The top three phyla of the
Crude protein content (%) 35.59
0.23a 29.29
0.95b bacteria community in the gut of T-Artemia were Proteobacteria
Fatty acid composition ( > 1%)
C14:0 1.54
0.78a 1.65
0.22a
C16:0 23.58
1.92a 22.94
3.06a Table 2
C16:1 1.29
0.53a 2.25
0.51a Comparison of diversity estimators of the bacteria communities of E-flocs and T-
C18:0 5.80
1.56a 4.91
0.36a flocs.
C18:1n9c 43.84
3.10a 42.09
1.74a
Sample OTU Ace Chao Shannon Simpson Coverage (%)
C18:1n7 2.02
1.70a 4.75
0.22b
C18:2n6 23.28
2.00a 20.21
0.19b E-flocs 179a
207 a
210a
2.15 a
0.38a
99.84a
C18:3n3 1.72
0.35a 1.28
0.33a T-flocs 162a 190a 197a 2.37a 0.26a 99.85a

Values are mean
standard deviation (n ¼ 3). Different superscripts in the same Values are mean
standard deviation (n ¼ 3). Different superscripts in the same
row denote significant differences among values. E-flocs: flocs produced with row denote significant differences among values. E-flocs: flocs produced with
solids waste from recirculating aquaculture system stocked European eel solids waste from recirculating aquaculture system stocked European eel
(Anguilla anguilla). T-flocs: flocs produced with solids waste from recirculating (Anguilla anguilla). T-flocs: flocs produced with solids waste from recirculating
aquaculture system stocked Nile tilapia (Oreochromis niloticus). aquaculture system stocked Nile tilapia (Oreochromis niloticus).

249
M. Yao et al. Aquaculture and Fisheries 3 (2018) 246–253

Fig. 2. Overlap of the two bacterial communities and phylogenetic classification at the genus level of the bacteria communities of T-flocs and E-flocs.

Table 3
Survival rate (%), individual length (mm), individual weight (mg) and biomass production (g/L) of E-Artemia and T-Artemia.
Groups Survival rate (%) Individual length (mm) Individual weight (mg) Biomass production (g/L)
a a a
E-Artemia 22
0.02 5.63
1.02 2.38
0.40 1.57
0.12a
T-Artemia 16
0.02b 5.90
1.92a 2.91
0.21a 1.40
0.08a

Table 4 bacteria community in the guts of T-Artemia and E-Artemia, and the
Fatty acid composition and crude protein content of E-Artemia and T-Artemia. genera in the T-Artemia gut and E-Artemia gut were significantly different
Fatty acids (%) E-Artemia T-Artemia from those in the bioflocs.
C14:0 0.80
0.07a 0.97
0.13a
C15:0 0.67
0.11a 0.74
0.13b 4. Discussion
C16:0 10.53
1.63a 12.13
0.75b
C16:1 4.77
0.78a 4.27
0.43a The objective of the current experiment was to compare the efficiency
C17:0 1.81
0.04a 0.91
0.12b of feeding two types of bioflocs to Artemia. The crude protein content
C18:0 5.94
0.88a 5.37
0.76a
C18:1n9t – 1.72
0.19b
(DW%) was within the range of the 23%–50% stated in the related re-
C18:1n9c 23.07
2.79a 35.39
1.20b ports (Avnimelech, 2012; Azim et al., 2007; Khanjani, Sajjadi, Alizadeh,
C18:1n7 11.21
3.43a 7.56
1.78b & Sourinejad, 2017). The crude protein content in E-flocs was higher
C18:2n6c 19.36
4.45a 16.43
1.61a than that of T-flocs. This result might be because the nitrogen content in
C20:0 4.28
2.12a 2.26
0.51b
European eel waste was higher than that of tilapia waste in the current
C18:3n6 1.80
0.25 –
C18:3n3 1.47
0.33a 0.87
0.12b experiment.
C20:4n6 3.54
2.49a 1.11
0.35b The crude protein contents of Artemia (70.01%
0.92% for E-Artemia
C20:5n3(EPA) 3.00
0.46a 1.57
0.19b and 65.63%
0.89% T-Artemia) were higher than those (40%–60%)
Crude protein content (%) 70.01
0.92a 65.63
0.89a reported in related studies (Anh, Van, Van, & Sorgeloos, 2009; Sorgeloos,
Values are mean
standard deviation (n ¼ 3). Different superscripts in the same 1989). This was perhaps because the crude protein content of the bioflocs
row denote significant differences among values. E-Artemia: Artemia fed on flocs was higher than the other food offered to Artemia, e.g., Chlorella
produced with solids waste from recirculating aquaculture system stocked Eu- (approximately 14%) (Luo et al., 2017). In our previous research, the
ropean eel (Anguilla anguilla). T-Artemia: Artemia fed on flocs produced with crude protein contents of the Artemia that were only fed flocs and Artemia
solids waste from recirculating aquaculture system stocked Nile tilapia (Oreo- fed flocs mixed with Chlorella were significantly higher than that of the
chromis niloticus). “-” means non-detected. Artemia fed only Chlorella (Luo et al., 2017). Docosahexaenoic acid
(DHA), which is one of the essential fatty acids, was not identified (Table
5). The low levels of DHA usually found in Artemia raise concerns by
(34.94%), cyanobacteria (31.07%) and Firmicutes (26.56%) (Fig. 3). 11 researchers (Cutts, Sawanboonchun, Mazorra de Quero, & Bell, 2006).
out of 183 genera had relative abundances higher than 1% for the gut of The previous study suggested that the DHA level in Artemia was partic-
T-Artemia. The bacteria genera in the T-flocs, E-flocs, T-Artemia gut, and ularly hard to increase through the using of any enrichment process
E-Artemia gut are shown in Table 5 in order to understand the bacteria (Hach
e & Plante, 2011).
communities among the two bioflocs and the two types of Artemia. Par- In addition to the essential nutrients in bioflocs, such as crude protein
vibaculum was the most dominant genus in T-flocs and E-flocs. At the and fatty acid, the presence of bacteria in bioflocs is believed to play a
genera level, similarity is relatively high between the bacteria commu- prominent role in the value of the bioflocs as a food source for aquatic
nity of E-flocs and T-flocs. The difference is more obvious between the animals. Bacterial enzymes in bioflocs were detected in many research

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Fig. 3. Overlap of the two bacterial communities and phylogenetic classification at the genus level of the gut bacteria communities of E-Artemia and T-Artemia.

Table 5 was reported in the current experiment and the related research (Crab,
Bacteria genera (abundance top 10) in T-flocs, E-flocs, and gut of T-Artemia gut Kochva, & Verstraete, 2009; Wei, Liao, & Wang, 2016).
and E-Artemia. Two types of bioflocs were dominated by cyanobacteria, Proteobac-
T-flocs Abundance E-flocs Abundance teria, and Bacteroidetes, consistent with the results of the related
(%) (%) research (Ferreira et al., 2015; Wei et al., 2016). cyanobacteria can grow
Parvibaculum 60.61 Parvibaculum 49.79 well under heterotrophic conditions and is characterized by long fila-
Propionibacteriaceae 6.69 Dietzia 8.42 mentous flattened cells (Baeza, Lopez, & Ehas, 2017). The Proteobacteria
norank detected is symbiotic in aquaculture (Sakami, Fujioka, & Shimoda,
Bradyrhizobium 3.93 Bradyrhizobium 6.14
2008). Proteobacteria removes organic matter, especially in wastewater
Sporosalibacterium 2.96 Sporosalibacterium 4.55
Thermomonas 2.45 Brevibacillus 3.51 treatment (Miura et al., 2007), and bioflocs aquaculture systems (Wei et
Brevibacillus 2.05 Salimesophilobacter 3.45 al., 2016). Similar to our results, it was reported that Bacteroidetes were
Bdellovibrio 1.92 mle1-27 3.14 dominant in both biofilm and granules in a sequencing batch biofilter
Carnobacterium 1.60 OPB35 soil group 2.70 reactor (De Sanctis, Di Iaconi, Lopez, & Rossetti, 2010).
Flavobacteriaceae 1.18 Microbulbifer 1.88
Brachybacterium 1.05 Arcobacter 1.53
The survival rate (16%–22%) was slightly lower than the previous
research (21.90%–46.90%) of Toi et al. (2013). The biomass production
T-Arteima Abundance E-Arteima Abundance
(1.40–1.57 g/L) in our experiment was within the range of 1.00–5.40 g/L
(%) (%)
in the previous research (Toi et al., 2013). This result may be due to the
Cyanobacteria 43.21 Parvibaculum 31.05 sizes of flocs (0.01–1.00 mm, not presented here) in the current experi-
Bacillus 8.61 Sneathiella 15.94
Marinobacter 7.09 Aequorivita 7.83
ment being too large for Artemia, especially younger individuals,
Shinella 4.87 Lachnospiraceae 6.40 decreasing the feeding efficiency. Artemia is a filter feeder of small food
Lactococcus 3.64 Microbulbifer 4.14 particles ranging 1–50 μm in size (D’Agostino, 1980). Biofloc size may
Aminobacter 2.81 Chryseobacterium 3.17 not only relate to the uptake potential of the biofloc by Artemia, but also
Pseudomonas 2.74 Azospira 2.89
the digestibility of the flocs as well as the nutritional value of the flocs (De
Caminicella 1.97 Holosporaceae 2.01
Sphingomonas 1.86 Terrisporobacter 1.92 Schryver, Crab, Defoirdt, Boon, & Verstraete, 2008). The result of Ekasari
Saccharibacteria 1.73 Armatimonadetes 1.13 et al. (2014) showed that the biofloc class of >100 μm contained the
highest levels of proteins (27.8%) and lipids (7.5%), whereas the biofloc
Values are mean
standard deviation (n ¼ 3). Different superscripts in the same
column denote significant differences among values. E-flocs: flocs produced with
of <48 μm seemed to be richest in essential amino acids. The biomass
solids waste from recirculating aquaculture system stocked European eel production can be improved by breaking the produced bioflocs into
(Anguilla anguilla). T-flocs: flocs produced with solids waste from recirculating different particle sizes according to the size of Artemia. Perhaps it would
aquaculture system stocked Nile tilapia (Oreochromis niloticus). E-Artemia: Arte- be better if the bioflocs were crushed or autoclaved to make them more
mia fed on flocs produced with solids waste from recirculating aquaculture sys- accessible for the nauplii to feed on. However, autoclaving or crushing
tem stocked European eel (Anguilla anguilla). T-Artemia: Artemia fed on flocs might cause changes in the structure of the bioflocs, destroying extra-
produced with solids waste from recirculating aquaculture system stocked Nile cellular polymers and cell wall material, which will destroy the healthy
tilapia (Oreochromis niloticus). probiotic. Consequently, if the bioflocs were not fed on time, they would
perish and pollute the water. Our experiment lasted for 19 days. We fed
studies (Luo et al., 2014; Xu & Pan, 2012). This was the main reason that the live bioflocs, without crushing or autoclaving the bioflocs before
the current experiment directly fed Artemia fresh bioflocs, to protect the feeding. This feeding strategy might have decreased the availability of
biological activities of the bioflocs. High bacteria diversity in bioflocs bioflocs to Artemia. More effective feeding techniques must be developed

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in the future. In addition, a full analysis would need to also assess the De Schryver, P., & Verstraete, W. (2009). Nitrogen removal from aquaculture pond water
by heterotrophic nitrogen assimilation in lab-scale sequencing batch reactors.
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0.89% for T-Artemia. The survival Fern
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