Growth, Productivity and Nutrient Uptake Rates of Ulva lactuca and Devaleraea mollis Co-Cultured with Atractoscion nobilis in a Land-Based Seawater Flow-Through Cascade IMTA System

Abstract

To advance environmentally friendly technologies in the aquaculture of Atractoscion nobilis, and simultaneously to diversify seafood production, a 79-day trial was conducted to assess the performance of Ulva lactuca and Devaleraea mollis cultured in the effluent from A. nobilis in a land-based integrated multi-trophic aquaculture (IMTA) system in southern California, USA. Water quality and performance of macroalgae were measured weekly. The impacted factors on the growth of macroalgae and nutrient uptake rate of macroalgae were assessed. The specific growth rate of juvenile A. nobilis was 0.47–0.52%/d. Total ammonia nitrogen in effluents of A. nobilis tanks ranged from 0.03 to 0.19 mg/L. Ulva lactuca and D. mollis achieved an average productivity of 24.53 and 14.40 g dry weight (DW)/m2/d. The average nitrogen content was 3.48 and 4.89% DW, and accordingly, the average nitrogen uptake rate was 0.88 and 0.71 g/m2/d, respectively. Temperature and nutrient concentration were key factors impacting macroalgae growth, and light intensity also impacted the growth of D. mollis. The high protein content of U. lactuca and D. mollis would make them good for use as human or animal food, or for use in other industries. Research on the interaction effects between seawater exchange rates and aeration rates on the performance and nutrient uptake rates of macroalgae will be conducted in future studies.

Full text

Citation: Huo, Y.; Elliott, M.S.;
Drawbridge, M. Growth, Productivity
and Nutrient Uptake Rates of Ulva
lactuca and Devaleraea mollis
Co-Cultured with Atractoscion nobilis
in a Land-Based Seawater
Flow-Through Cascade IMTA System.
Fishes 2024,9, 417. https://doi.org/
10.3390/fishes9100417
Academic Editor: Ronaldo
Olivera Cavalli
Received: 12 September 2024
Revised: 11 October 2024
Accepted: 17 October 2024
Published: 19 October 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
fishes
Article
Growth, Productivity and Nutrient Uptake Rates of Ulva lactuca
and Devaleraea mollis Co-Cultured with Atractoscion nobilis in a
Land-Based Seawater Flow-Through Cascade IMTA System
Yuanzi Huo, Matthew S. Elliott and Mark Drawbridge *
Hubbs-SeaWorld Research Institute, San Diego, CA 92109, USA; yhuo@hswri.org (Y.H.);
melliott@hswri.org (M.S.E.)
*Correspondence: mdrawbridge@hswri.org; Tel.: +1-619-226-3870; Fax: +1-619-226-3944
Abstract: To advance environmentally friendly technologies in the aquaculture of Atractoscion no-
bilis, and simultaneously to diversify seafood production, a 79-day trial was conducted to assess
the performance of Ulva lactuca and Devaleraea mollis cultured in the effluent from A. nobilis in a
land-based integrated multi-trophic aquaculture (IMTA) system in southern California, USA. Water
quality and performance of macroalgae were measured weekly. The impacted factors on the growth
of macroalgae and nutrient uptake rate of macroalgae were assessed. The specific growth rate of
juvenile A. nobilis was 0.47–0.52%/d. Total ammonia nitrogen in effluents of A. nobilis tanks ranged
from 0.03 to 0.19 mg/L. Ulva lactuca and D. mollis achieved an average productivity of 24.53 and
14.40 g dry weight (DW)/m
2
/d. The average nitrogen content was 3.48 and 4.89% DW, and ac-
cordingly, the average nitrogen uptake rate was 0.88 and 0.71 g/m
2
/d, respectively. Temperature
and nutrient concentration were key factors impacting macroalgae growth, and light intensity also
impacted the growth of D. mollis. The high protein content of U. lactuca and D. mollis would make
them good for use as human or animal food, or for use in other industries. Research on the interaction
effects between seawater exchange rates and aeration rates on the performance and nutrient uptake
rates of macroalgae will be conducted in future studies.
Keywords: biofilter; nutrient dynamics; eutrophication; bioremediation; mariculture
Key Contribution: Ulva lactuca and Devaleraea mollis achieved high productivities; nutritional compo-
sition and nutrient uptake rates when co-cultured with Atractoscion nobilis in a land-based seawater
flow-through cascade IMTA system. Increased nutrient concentrations are expected to improve the
performance of macroalgae co-cultured with A. nobilis.
1. Introduction
In the next 30 years, global seafood demand is expected to grow 30%, and aquaculture
is expected to meet nearly all the increased global demand [
1
]. To successfully expand
seafood production, aquaculturists must continually try to meet high sustainability stan-
dards expected by the public [
2
]. For aquaculture producers, access to seawater and coastal
space is very limited and difficult to acquire in most cases [
3
]. Any space and water that is
available needs to be maximized in its use. Also, the seafood marketplace is highly compet-
itive and one’s ability to diversify product lines to include multiple, high-value products,
as well as gain operational efficiencies, will be greatly beneficial [
4
]. Diversified product
lines will also help buffer against production failures for any given single species [5].
Waste (e.g., solids and nutrients) produced from mono-cultured fed species can also be
removed by a combination of assimilatory and dissimilatory processes, mediated by pho-
totrophic and heterotrophic organisms. This modern form of polyculture is called integrated
multi-trophic aquaculture (IMTA), in which phototrophic and heterotrophic organisms
are intentionally used to remove waste from the system, while concurrently diversifying
Fishes 2024,9, 417. https://doi.org/10.3390/fishes9100417 https://www.mdpi.com/journal/fishes

Fishes 2024,9, 417 2 of 16
production [
6
]. Among phototrophic organisms used in IMTA systems, macroalgae is
demonstrated to be a good biological filter to remove nutrients from the water column
and re-oxygenate the culture water, thereby providing a positive environment for cultured
animals [
6
,
7
]. Many studies have already been conducted in open-water IMTA systems that
integrated macroalgae with fish, bivalves, and sea cucumbers [
8
–
11
]. In recent years, much
attention has been focused on land-based IMTA systems, and various models have been
proposed that mostly seek to integrate macroalgae with finfish, shrimp, abalone, bivalves,
and echinoderms [
11
–
18
]. In these studies, macroalgae showed great efficiencies in biomass
production and nutrient waste uptake. Interest in this topic is growing rapidly, and more
research publications should be forthcoming.
White seabass Atractoscion nobilis is the target of a commercial fishery within its range
that extends from central California, USA to Baja California, Mexico [
19
]. Atractoscion nobilis
has numerous aquaculture characteristics desirable for commercialization, and after years
of research, the hatchery rearing protocols for A. nobilis from spawning through fingerling
production are now among the most advanced for the intensive hatchery production of
a marine fish species in the United States [
19
]. To advance environmentally friendly tech-
nologies in the culture of A. nobilis, and simultaneously diversify seafood production, it
is worthwhile to evaluate the integration of A. nobilis with low trophic organisms. Draw-
bridge et al. [
20
] reported that the growth, productivity, nutritional quality, and nutrient
waste removal rate of Ulva lactuca were improved when they were integrated into an IMTA
system co-culturing with A. nobilis in southern California, USA.
Ulva species have been identified as an ideal biofilter due to their strong capacity for ni-
trogen and phosphorus uptake and robust resilience to environmental
changes [
21
,
22
]. Additionally, Ulva has wide applications in human food, animal feed, bio-
fuel, and medicine [
18
,
23
,
24
]. In addition to Ulva species, red macroalgae species have also
been used in IMTA systems. Devaleraea mollis is a low-intertidal to subtidal red macroalgae
species that is native to waters of the northeastern Pacific Ocean from Alaska to southern
California and around Asia. Due to its high nutritional quality and growth rate [
25
,
26
], D.
mollis has been used as a biofilter and feed in abalone polyculture as well as for human
consumption [13,27,28].
Until now, except for the study conducted by Drawbridge et al. [
20
] using Ulva as
a nutrient biofilter in three-week trials, no other studies on A. nobilis IMTA have been
conducted for a relatively long period, and also no studies have co-cultured D. mollis with
finfish in IMTA systems. In this study, A. nobilis was integrated with U. lactuca and D.
mollis in a land-based flow-through cascade IMTA system. A 79-day trial was conducted to
evaluate the growth, productivity, nutritional composition, and nutrient removal rates of U.
lactuca and D. mollis cultivated in the effluent from A. nobilis culture tanks, and to confirm
what environmental parameters were the controlling factors to impact the performance of
U. lactuca and D. mollis. Based on the results of the present study, future worthwhile studies
were suggested. The results of the present study can be used to help establish an efficient
IMTA system integrating A. nobilis with multi-trophic level organisms that increases and
diversifies farm production while reducing nutrient discharge into coastal areas.
2. Materials and Methods
2.1. The Cascade IMTA System Design
A flow-through seawater cascade IMTA system was designed as shown in Figure 1.
Four tiers were constructed in a stepped manner to support black polyethylene tanks
with a working volume of 700 L (height: 70 cm; surface diameter: 60 cm; floor diameter:
52 cm) and a surface area of 1.09 m
2
each. The tiered tanks allowed seawater to cascade
by gravity from the first to the last prior to discharge such that water was only pumped
once to the system. Each tiered unit of four tanks was set up in triplicate rows for a total
of 12 tanks. Three tanks on the first tier were used to hold A. nobilis as the primary “fed”
species. Incoming ambient seawater was pumped from Aqua Hedionda Lagoon through
sand filters into a holding tank that allowed for degassing and also ensured uninterrupted

Fishes 2024,9, 417 3 of 16
water flow to the fish tanks, and then seawater was pumped into each fish tank using a 1/3
HP pump (Sequence 1000 External, Colorado Springs, CO, USA) with ball valves at each
tank inlet to control flow rate. The water inlet to each fish tank consisted of a horizontal
spray bar (3.8 cm) to provide additional degassing and directional water currents. Each fish
tank was also supplied with aeration using air stones and a standby supply of pure oxygen
if needed. The water was discharged through a center standpipe that set the water level of
the tank. A PVC sleeve (15 cm diameter) with slits at the bottom was placed around the
5 cm standpipe to facilitate self-cleaning, whereby a suction was created using airstones
inside the sleeve as an air-lift. This drain design, combined with circular water currents
in the tank, effectively pulled settled solids up and out of the tank into Tier-2. Tanks on
Tier-2 were designed to culture invertebrates such as sea cucumbers, abalone, sea urchins,
polychaetes, and filter feeders (e.g., oysters, mussels), but that was not part of this study.
The seawater from Tier-2 tanks was directed out from the side of the tank at the surface
into Tier-3 tanks which were used to support the tumbled-culture of seaweed as nutrient
biofilters in this system. Each seaweed tank had a 5 cm side drain at a height of 0.71 m. The
drain pipe was connected to a 0.56 m long 5 cm mesh tube (4 mm) on the interior of the
tank to keep seaweed from going out the drain. An air ring was installed at the bottom of
the standpipe to provide central aeration at a rate of 25 L/min to drive seaweeds up and
around the entire tank equally in a vertical circular motion. The seawater from each tank
on Tier-3 entered each tank on Tier-4 through a side drain similar to that in between Tier-2
and Tier-3. The tanks on the fourth tier were also designed to support the “second stage”
seaweed cultivation as biofilters, which had the same structure design as the tanks on the
third tier. The seawater effluent from Tier-4 was also screened and exited the side of the
tanks before being discharged into Aqua Hedionda Lagoon.
Fishes 2024, 9, x FOR PEER REVIEW 3 of 16
from the rst to the last prior to discharge such that water was only pumped once to the
system. Each tiered unit of four tanks was set up in triplicate rows for a total of 12 tanks.
Three tanks on the rst tier were used to hold A. nobilis as the primary “fed” species. In-
coming ambient seawater was pumped from Aqua Hedionda Lagoon through sand lters
into a holding tank that allowed for degassing and also ensured uninterrupted water ow
to the sh tanks, and then seawater was pumped into each sh tank using a 1/3 HP pump
(Sequence 1000 External, USA) with ball valves at each tank inlet to control ow rate. The
water inlet to each sh tank consisted of a horizontal spray bar (3.8 cm) to provide addi-
tional degassing and directional water currents. Each sh tank was also supplied with
aeration using air stones and a standby supply of pure oxygen if needed. The water was
discharged through a center standpipe that set the water level of the tank. A PVC sleeve
(15 cm diameter) with slits at the boom was placed around the 5 cm standpipe to facili-
tate self-cleaning, whereby a suction was created using airstones inside the sleeve as an
air-lift. This drain design, combined with circular water currents in the tank, eectively
pulled seled solids up and out of the tank into Tier-2. Tanks on Tier-2 were designed to
culture invertebrates such as sea cucumbers, abalone, sea urchins, polychaetes, and lter
feeders (e.g., oysters, mussels), but that was not part of this study. The seawater from Tier-
2 tanks was directed out from the side of the tank at the surface into Tier-3 tanks which
were used to support the tumbled-culture of seaweed as nutrient biolters in this system.
Each seaweed tank had a 5 cm side drain at a height of 0.71 m. The drain pipe was con-
nected to a 0.56 m long 5 cm mesh tube (4 mm) on the interior of the tank to keep seaweed
from going out the drain. An air ring was installed at the boom of the standpipe to pro-
vide central aeration at a rate of 25 L/min to drive seaweeds up and around the entire tank
equally in a vertical circular motion. The seawater from each tank on Tier-3 entered each
tank on Tier-4 through a side drain similar to that in between Tier-2 and Tier-3. The tanks
on the fourth tier were also designed to support the “second stage” seaweed cultivation
as biolters, which had the same structure design as the tanks on the third tier. The sea-
water euent from Tier-4 was also screened and exited the side of the tanks before being
discharged into Aqua Hedionda Lagoon.
Figure 1. A seawater ow-through cascade IMTA system containing four tiers for integrating fed
sh species (A), invertebrates, and two-stage seaweeds (B). The working volume of all tanks was
the same as 700 L with a surface area of 1.09 m2 each.
2.2. Fish and Seaweed Sourcing
White seabass A. nobilis were spawned and reared at the Hubbs-SeaWorld Research
Institute (HSWRI) marine sh hatchery (Carlsbad, California, CA, USA). Devaleraea mollis
was provided by The Cultured Abalone Farm (Goleta, California, CA, USA), and U. lactuca
was provided by the San Diego State University’s Coastal Marine Institute Laboratory
(San Diego, California, CA, USA). Devaleraea mollis and U. lactuca were initially stocked
into 175 L cone-boom tanks and a larger 3300 L holding tank supplied with euent from
A. nobilis raceway at an exchange rate of 63 vol./day under ambient daylight conditions.
Devaleraea mollis and U. lactuca were allowed to grow in these tanks until the start of this
experiment. In this study, no invertebrates were integrated into this IMTA system. Rather,
Fish
tanks
Invertebrate
tanks
Seaweed
tanks
A
B
Invertebrate and
seaweed tanks
shown in B.
Figure 1. A seawater flow-through cascade IMTA system containing four tiers for integrating fed
fish species (A), invertebrates, and two-stage seaweeds (B). The working volume of all tanks was the
same as 700 L with a surface area of 1.09 m2 each.
2.2. Fish and Seaweed Sourcing
White seabass A. nobilis were spawned and reared at the Hubbs-SeaWorld Research
Institute (HSWRI) marine fish hatchery (Carlsbad, California, CA, USA). Devaleraea mollis
was provided by The Cultured Abalone Farm (Goleta, California, CA, USA), and U. lactuca
was provided by the San Diego State University’s Coastal Marine Institute Laboratory (San
Diego, California, CA, USA). Devaleraea mollis and U. lactuca were initially stocked into
175 L cone-bottom tanks and a larger 3300 L holding tank supplied with effluent from
A. nobilis raceway at an exchange rate of 63 vol./day under ambient daylight conditions.
Devaleraea mollis and U. lactuca were allowed to grow in these tanks until the start of
this experiment. In this study, no invertebrates were integrated into this IMTA system.
Rather, the three Tier-2 invertebrate tanks served as “sumps” to receive seawater containing
nutrient and solid wastes from A. nobilis tanks (Tier-1). Seawater entered into the seaweed
tanks by gravity from the top of Tier-2 and solid wastes that settled to the bottom of Tier-2
were siphoned out at regular intervals so they did not move into downstream tiers.

Fishes 2024,9, 417 4 of 16
2.3. Experimental Design
2.3.1. IMTA Fish Component
Juvenile A. nobilis with an average body weight of 134.7
±
16.0 g were stocked at a
density of 30 kg/m
3
in each replicate Tier-1 tank. Individual total and standard lengths
were measured to the nearest 1.0 mm and wet body weights to the nearest 1.0 g at days
0, 47, and 79. Fish were anesthetized using tricaine mesylate (MS-222) at 75 mg/L during
the weighing and stocking density setting-up process [
29
]. Fish were fed a commercial
growout diet (Skretting 4.0 mm Marine mix with a proximate composition of 46% protein
and 12% fat) to satiation by hand twice daily (1–3% body weight per day) between 8:00 and
10:00 and between 14:00 and 16:00. Any mortalities were removed daily and general health
status was monitored during feeding. To compensate for fish growth, the fish density was
reset to 30 kg/m
3
on 8th May by removing an appropriate number of fish from each tank.
This effectively divided the study period into two phases: Mar 23 to 8 May (47 days), and 8
May to 8 June (32 days). The seawater exchange rate was set to 63 vol./day (2100 L/kg)
according to our previous study [
20
]. Each tank of A. nobilis had a 30% shade cover on top
to keep fish from jumping out as well as a shade canopy above all the fish tanks. Average
temperature ranged from 14.3
◦
C to 19.7
◦
C, dissolved oxygen (DO) was in the range of
8.08–10.55 mg/L, and photoperiod was 12 Light:12 Dark.
2.3.2. IMTA Seaweed Component
After rinsing with filtered seawater, U. lactuca was stocked at an initial density of 1 kg
wet weight/m
2
according to our previous studies [
6
,
20
], and D. mollis was stocked at the
initial density of 4 kg wet weight/m
2
based on other references [
30
,
31
]. Devaleraea mollis
tanks were covered with standard 60% nursery shade cloth to control epiphytes, which
was verified using a light meter (Extech 407026, Taibei, Taiwan). During the trial period, D.
mollis and U. lactuca were harvested weekly and reset to their respective initial stocking
densities mentioned above. To determine biomass, the seaweed was shaken gently in a
basket to remove residual seawater and then it was allowed to air dry for 5 min before
weighing to the nearest 1.0 g. For tissue analyses, 250 g sub-samples of each species were
taken at the beginning of the trial, at each weekly sampling day, and at the end of the
trial. Sub-samples were rinsed thoroughly with filtered seawater and then deionized water
several times, and wiped with tissue paper before being dried in an oven at 60
◦
C for 48 h
until a constant weight was achieved. Dried sub-samples were stored inside an oven at
60 ◦C until chemical composition was measured.
2.4. Data Collection
2.4.1. Environmental and Water Quality
Light and temperature levels at the surface of the water in D. mollis and U. lactuca tanks
were measured and logged every 15 min using HOBO Pendant MX2202 Temperature/Light
Data Logger (Onset, Bourne, MA, USA). Temperature was also logged separately in ambient
air and in the A. nobilis tanks. DO and pH were measured twice daily (8:00 and 14:00) using
a Hach HQ40d hand held multi-meter (Hach, Loveland, CO, USA) and a Pinpoint pH meter
(American Marine, Ridgefield, CT, USA). Seawater samples were collected from the influent
of the A. nobilis tanks, and influents and effluents of each seaweed tank once each week at
13:00 on sampling dates to measure the total ammonia nitrogen (TAN), nitrite (NO
2
-N) and
nitrate (NO
3
-N), and phosphate (PO
4
-P) concentrations. TAN, NO
2
-N, NO
3
-N, and PO
4
-P
concentration analyses were made in duplicates using a Hach DR 6000 spectrophotometer
after filtering the sample through a 0.45 µm CA membrane filter.
2.4.2. Biological and Biochemical Measures
The specific growth rate (SGR) of A. nobilis was calculated based on the weight dif-
ference between the initial stocking density resetting day, and the end of the trial. Food
conversion rate (FCR) was calculated by summing up the amount of food fed during the
trial and dividing by the increase in population biomass. The specific growth rate (SGR)

Fishes 2024,9, 417 5 of 16
and productivity of D. mollis and U. lactuca were assessed weekly by measuring the differ-
ence of wet-weight biomass. Seaweed tissue nitrogen and carbon were measured using a
Costech 4010 gas chromatography elemental analyzer. The protein content of D. mollis and
U. lactuca was determined by multiplying the nitrogen content by 6.25 [32].
2.4.3. Parameter Calculation
Nutrient removal efficiency and rate by D. mollis and U. lactuca was based on the
difference of nutrient concentrations between influents and effluents of seaweed tanks, and
tissue nitrogen, carbon content, and productivity. All parameters were calculated based on
following equations:
Specific growth rate (SGR, %/day) =100 ×(lnWf−lnWi)/t(1)
Food conversion rate (FCR) for A. nobilis =Feed Given/Weight Gain (2)
Productivity (g dry weight (DW)/m2/d) of seaweed = DW×(Wf−Wi)/a/t(3)
Nutrient removal efficiency (%) = [(Cin −Cout)/Cin]×100 (4)
Nutrient uptake rate (g/m2/day) = Ctissue (or Ntissue)×Productivity (5)
where W
f
was the final wet weight, W
i
was initial wet weight, and twas the experimen-
tal time (days); DW was the percentage dry weight of seaweed, awas the surface area
of tank (m
2
), C
in
was the nutrient concentration in influents, and C
out
was the nutrient
concentrations in effluents; C
tissue
and N
tissue
was the seaweed tissue carbon and nitrogen
concentrations.
2.5. Data Analysis
Data were expressed as mean
±
standard deviation. Data analysis was performed
using JMP Pro software (version 15.0, SAS Institute). Tests of homogeneity of variance
were conducted and percentage data, such as nutrient removal efficiencies, were arsine
transformed for normalization before analysis. ANOVA analysis and Pearson correlations
were used in this study for data analyses. Tukey’s least significant difference (LSD) was
used to make post hoc comparisons between different combinations. Differences were
considered significant at p< 0.05.
3. Results
3.1. Environmental Conditions
The average seawater temperature ranged from 14.3
◦
C to 19.7
◦
C with a minimum of
12.6
◦
C and maximum of 25.8
◦
C during the experimental period (Figure 2left). The average
temperature increased gradually from 15.1
◦
C on 17 April to 19.6
◦
C at the beginning of
June when the trial was finished. The PAR level varied significantly between days during
the experimental period. Because of cloudy and raining days, the median PAR level of
22.4–127.8
µ
mol/m
2
s was significantly lower during 3–5 April, 20 April–1 May and 26–28
May compared with other experimental days when the median PAR level ranged from
228.7 µmol/m2s to 2260.4 µmol/m/2s (Figure 2right).
The average pH ranged from 7.75 to 8.16 in the influents of the seaweed tanks with the
average value of 7.93
±
0.03, and the average pH ranged from 7.81 to 8.55 in the effluents of
seaweed tanks with the average value of 8.01
±
0.05 (Figure 3). The seaweed tank influent
was the same as the fish tank effluent.

Fishes 2024,9, 417 6 of 16
Fishes 2024, 9, x FOR PEER REVIEW 6 of 16
12
14
16
18
20
22
24
26
Temperature (°C)
25%~75%
Range within 1.5IQR
Median Line
Mean
Outliers
25-Mar 15-Apr 1-May 15-May 6-Jun
1-Apr 31-May
0
2
4
6
25%~75%
Range within 1.5IQR
Median Line
Mean
Outliers
PAR (´103 mm/m2/s)
25-Mar1-Apr 15-Apr 1-May 15-May 31-May6-Jun
Figure 2. The change of daily seawater temperature (left) and light intensity (right) at the surface of
the seaweed tanks throughout the experimental period from 25 March to 6 June (n = 3). The values
25–75% contain the middle 50% of the data. IQR means the interquartile range, and 1.5IQR means
1.5 points below the lower bound quartile or above the upper bound quartile is an outlier.
The average pH ranged from 7.75 to 8.16 in the inuents of the seaweed tanks with
the average value of 7.93 ± 0.03, and the average pH ranged from 7.81 to 8.55 in the eu-
ents of seaweed tanks with the average value of 8.01 ± 0.05 (Figure 3). The seaweed tank
inuent was the same as the sh tank euent.
Mar 21 Apr 4 Apr 18 May 2 May 16 May 30
7.8
8.0
8.7
9.0
pH
Effluent
Influent
Figure 3. The change of monitored average pH values (measured twice daily at 8:00 and 14:00, re-
spectively) in the inuents and euents of seaweed tanks throughout the experimental period
where the seaweed tank inuent is the same as the white seabass tank euent (n = 3).
3.2. Atractoscion Nobilis Growth and Food Conversion Rate (FCR)
The weight gain of white seabass increased from 120.4 ± 4.4 g to 149.0 ± 3.0 g with the
increasing percentage of 23.75% and from 149.0 ± 3.0 g to 188.1 ± 3.0 g with the increasing
percentage of 26.24% in Phase 1 and 2, respectively, and consequently, the calculated SGR
of white seabass was 0.47 ± 0.05%/d and 0.52 ± 0.05%/d and the FCR was 2.05 ± 0.27 and
1.64 ± 0.20 in Phase 1, and in Phase 2, respectively, but no signicant dierence was de-
tected in SGR (p = 0.37) and FCR (p = 0.10) between the two phases (Table 1).
Table 1. The phase period, initial number with initial average weight (g), feeding rate (% body
weight), nal average weight (g), growth rate (SGR, %/day) and food conversion ratio (FCR) of white
seabass (Atractoscion nobilis) in Phase 1 and Phase 2 during the experimental period (n = 3).
Phase
Period
Initial Number
(No.)
Initial Average
Weight (G)
Feeding Rate (%
Body Weight)
Final Average
Weight (G)
SGR (%/Day)
FCR
1
23 March–8 May
175 ± 6
120.4 ± 4.4
1.07 ± 0.002
149.0 ± 3.0
0.47 ± 0.05
2.05 ± 0.27
Figure 2. The change of daily seawater temperature (left) and light intensity (right) at the surface of
the seaweed tanks throughout the experimental period from 25 March to 6 June (n = 3). The values
25–75% contain the middle 50% of the data. IQR means the interquartile range, and 1.5IQR means
1.5 points below the lower bound quartile or above the upper bound quartile is an outlier.
Fishes 2024, 9, x FOR PEER REVIEW 6 of 16
12
14
16
18
20
22
24
26
Temperature (°C)
25%~75%
Range within 1.5IQR
Median Line
Mean
Outliers
25-Mar 15-Apr 1-May 15-May 6-Jun
1-Apr 31-May
0
2
4
6
25%~75%
Range within 1.5IQR
Median Line
Mean
Outliers
PAR (´103 mm/m2/s)
25-Mar1-Apr 15-Apr 1-May 15-May 31-May6-Jun
Figure 2. The change of daily seawater temperature (left) and light intensity (right) at the surface of
the seaweed tanks throughout the experimental period from 25 March to 6 June (n = 3). The values
25–75% contain the middle 50% of the data. IQR means the interquartile range, and 1.5IQR means
1.5 points below the lower bound quartile or above the upper bound quartile is an outlier.
The average pH ranged from 7.75 to 8.16 in the inuents of the seaweed tanks with
the average value of 7.93 ± 0.03, and the average pH ranged from 7.81 to 8.55 in the eu-
ents of seaweed tanks with the average value of 8.01 ± 0.05 (Figure 3). The seaweed tank
inuent was the same as the sh tank euent.
Mar 21 Apr 4 Apr 18 May 2 May 16 May 30
7.8
8.0
8.7
9.0
pH
Effluent
Influent
Figure 3. The change of monitored average pH values (measured twice daily at 8:00 and 14:00, re-
spectively) in the inuents and euents of seaweed tanks throughout the experimental period
where the seaweed tank inuent is the same as the white seabass tank euent (n = 3).
3.2. Atractoscion Nobilis Growth and Food Conversion Rate (FCR)
The weight gain of white seabass increased from 120.4 ± 4.4 g to 149.0 ± 3.0 g with the
increasing percentage of 23.75% and from 149.0 ± 3.0 g to 188.1 ± 3.0 g with the increasing
percentage of 26.24% in Phase 1 and 2, respectively, and consequently, the calculated SGR
of white seabass was 0.47 ± 0.05%/d and 0.52 ± 0.05%/d and the FCR was 2.05 ± 0.27 and
1.64 ± 0.20 in Phase 1, and in Phase 2, respectively, but no signicant dierence was de-
tected in SGR (p = 0.37) and FCR (p = 0.10) between the two phases (Table 1).
Table 1. The phase period, initial number with initial average weight (g), feeding rate (% body
weight), nal average weight (g), growth rate (SGR, %/day) and food conversion ratio (FCR) of white
seabass (Atractoscion nobilis) in Phase 1 and Phase 2 during the experimental period (n = 3).
Phase
Period
Initial Number
(No.)
Initial Average
Weight (G)
Feeding Rate (%
Body Weight)
Final Average
Weight (G)
SGR (%/Day)
FCR
1
23 March–8 May
175 ± 6
120.4 ± 4.4
1.07 ± 0.002
149.0 ± 3.0
0.47 ± 0.05
2.05 ± 0.27
Figure 3. The change of monitored average pH values (measured twice daily at 8:00 and 14:00,
respectively) in the influents and effluents of seaweed tanks throughout the experimental period
where the seaweed tank influent is the same as the white seabass tank effluent (n = 3).
3.2. Atractoscion Nobilis Growth and Food Conversion Rate (FCR)
The weight gain of white seabass increased from 120.4
±
4.4 g to 149.0
±
3.0 g with the
increasing percentage of 23.75% and from 149.0
±
3.0 g to 188.1
±
3.0 g with the increasing
percentage of 26.24% in Phase 1 and 2, respectively, and consequently, the calculated SGR
of white seabass was 0.47
±
0.05%/d and 0.52
±
0.05%/d and the FCR was 2.05
±
0.27
and 1.64
±
0.20 in Phase 1, and in Phase 2, respectively, but no significant difference was
detected in SGR (p= 0.37) and FCR (p= 0.10) between the two phases (Table 1).
Table 1. The phase period, initial number with initial average weight (g), feeding rate (% body
weight), final average weight (g), growth rate (SGR, %/day) and food conversion ratio (FCR) of white
seabass (Atractoscion nobilis) in Phase 1 and Phase 2 during the experimental period (n = 3).
Phase Period
Initial Number
(No.)
Initial Average
Weight (G)
Feeding Rate (%
Body Weight)
Final Average
Weight (G)
SGR (%/Day)
FCR
123 March–8
May 175 ±6 120.4 ±4.4 1.07 ±0.002 149.0 ±3.0 0.47 ±0.05
2.05
±
0.27
2
8 May–8 June
142 ±3 149.0 ±3.0 1.42 ±0.002 188.1 ±3.0 0.52 ±0.05
1.64
±
0.20

Fishes 2024,9, 417 7 of 16
3.3. Growth and Productivity of Devaleraea Mollis and Ulva lactuca
The SGR ranged from 0.28 to 5.27%/d and 7.38 to 19.22%/d for D. mollis and U.
lactuca with averages of 2.93
±
1.69%/d and 12.95
±
4.68%/d, respectively (Figure 4A).
The productivity was in the range of 1.23–25.95 g DW/m
2
d and 11.31–47.52 g DW/m
2
d for
D. mollis and U. lactuca with average values of 14.40
±
9.09 g DW/m
2
d and 24.53
±
15.34 g
DW/m
2
d, respectively (Figure 4B). The SGR of U. lactuca was significantly higher than that
of D. mollis (p< 0.01), and the productivity showed no difference between U. lactuca and D.
mollis (p= 0.059).
Fishes 2024, 9, x FOR PEER REVIEW 7 of 16
2
8 May–8 June
142 ± 3
149.0 ± 3.0
1.42 ± 0.002
188.1 ± 3.0
0.52 ± 0.05
1.64 ± 0.20
3.3. Growth and Productivity of Devaleraea Mollis and Ulva lactuca
The SGR ranged from 0.28 to 5.27%/d and 7.38 to 19.22%/d for D. mollis and U. lactuca
with averages of 2.93 ± 1.69%/d and 12.95 ± 4.68%/d, respectively (Figure 4A). The produc-
tivity was in the range of 1.23–25.95 g DW/m2d and 11.31–47.52 g DW/m2d for D. mollis
and U. lactuca with average values of 14.40 ± 9.09 g DW/m2d and 24.53 ± 15.34 g DW/m2d,
respectively (Figure 4B). The SGR of U. lactuca was signicantly higher than that of D.
mollis (p < 0.01), and the productivity showed no dierence between U. lactuca and D. mol-
lis (p = 0.059).
Devaleraea mollis Ulva lactuca
0
2
4
6
8
10
12
14
16
18
(SGR, %/day)
A
Devaleraea mollis Ulva lactuca
0
5
10
15
20
25
30
35
40
Productivity (gDW/m2/day)
B
0
2
4
6
8
10
12
14
16
18
20 Devaleraea mollis
Ulva lactuca
SGR (%/day)
Productivity (gDW/m2/day)
C
D
30-Mar
6-Apr
13-Apr
20-Apr
27-Apr
4-May
11-May
18-May
25-May
1-Jun
7-Jun
0
10
20
30
40
50
Figure 4. The average growth rate (SGR, %/day) (A) and productivity (g DW/m2d) (B), and variation
of growth rate (SGR, %/day) (C) and productivity (g DW/m2d) (D) of Devaleraea mollis and Ulva
lactuca cultivated with the euent from A. nobilis tanks.
The results of correlation analysis between the SGR of D. mollis and U. lactuca and
environmental factors is shown in Figure 5. The results showed that no relationship ex-
isted between TAN concentrations and the growth of D. mollis and U. lactuca, but it
showed negative relationships between the growth of D. mollis and U. lactuca and PO4-P
concentrations. The SGR of D. mollis and U. lactuca showed a negative relationship with
the pH. The SGR of D. mollis was negatively correlated to temperature, while the SGR of
U. lactuca was positively correlated with temperature. Also, the SGR of U. lactuca showed
a moderate positive relationship with NO3-N concentrations.
Figure 4. The average growth rate (SGR, %/day) (A) and productivity (g DW/m
2
d) (B), and variation
of growth rate (SGR, %/day) (C) and productivity (g DW/m
2
d) (D) of Devaleraea mollis and Ulva
lactuca cultivated with the effluent from A. nobilis tanks.
The results of correlation analysis between the SGR of D. mollis and U. lactuca and
environmental factors is shown in Figure 5. The results showed that no relationship
existed between TAN concentrations and the growth of D. mollis and U. lactuca, but it
showed negative relationships between the growth of D. mollis and U. lactuca and PO
4
-P
concentrations. The SGR of D. mollis and U. lactuca showed a negative relationship with the
pH. The SGR of D. mollis was negatively correlated to temperature, while the SGR of U.
lactuca was positively correlated with temperature. Also, the SGR of U. lactuca showed a
moderate positive relationship with NO3-N concentrations.

Fishes 2024,9, 417 8 of 16
Fishes 2024, 9, x FOR PEER REVIEW 8 of 16
Growth
TAN
NO3-N
NO2-N
PO4-P
N/PpH T
M L
MD L
Growth
TAN
NO3-N
NO2-N
PO4-P
N/P
pH
T
M L
MD L
0.031
−0.084−0.83
0.38 0.36−0.35
−0.54−0.60 0.70−0.061
0.49 0.73−0.560.090−0.88
0.35−0.36 0.19−0.81 0.81
−0.62 0.64−0.310.014 0.14 0.13−0.38
0.56−0.045−0.00610.34 −0.35 0.21 0.61−0.52
0.15 0.16−0.26 0.20−0.35 0.18 0.46−0.190.85
0.78
−1
−0.8
−0.6
−0.4
−0.2
0
0.2
0.4
0.6
0.8
1
Devaleraea mollis
Growth
TAN
NO3-N
NO2-N
PO4-P
N/PpH T
M L
MD L
Growth
TAN
NO3-N
NO2-N
PO4-P
N/P
pH
T
M L
MD L
2.3E−4
0.36−0.76
0.067 0.33−0.40
−0.58 0.50 0.048
0.64−0.26−0.085−0.88
−0.39 0.36−0.200.067−0.84 0.83
0.51 0.64−0.28−0.00540.11 0.15 −0.32
0.12−0.082−0.19 0.43−0.028−0.22 0.22−0.46
0.097 0.12−0.35 0.28−0.14−0.0980.24−0.20 0.86 −1
−0.8
−0.6
−0.4
−0.2
0
0.2
0.4
0.6
0.8
1
Ulva lactuca
0.30
−0.60
Figure 5. Correlation analysis between the growth rate of Devaleraea mollis and Ulva lactuca, and
environmental factors. Red circles indicate a positive correlation, and blue circles indicate a negative
correlation. Numbers in each cell represent the correlation coecient. Note: M L indicates mean
light intensity; MD L indicates median light intensity.
3.4. Nutrient Concentration and Removal Eciency
The TAN, NO3-N, NO2-N, and PO4-P concentrations in the inuents of seaweed tanks
varied greatly among sampling dates (Figure 6). Based on the dierence between inuent
and euent measurements, the nutrient removal eciency was 37.27 ± 29.25% and 44.50
± 30.70% for TAN; 56.67 ± 34.34% and 65.69 ± 31.81% for NO3-N; 29.67 ± 12.61% and 22.92
± 36.67% for NO2-N; and 50.95 ± 32.47% and 44.26 ± 17.74% for PO4-P by D. mollis and U.
lactuca, respectively. The removal eciency of each nutrient was not signicantly dierent
between D. mollis and U. lactuca (p = 0.64–0.88).
0.00
0.05
0.10
0.15
0.20
0.25
0.0
0.2
0.4
0.6
0.8
1.0
29-Mar
5-Apr
12-Apr
19-Apr
26-Apr
3-May
17-May
24-May
31-May
0.000
0.002
0.004
0.006 C
29-Mar
5-Apr
12-Apr
19-Apr
26-Apr
3-May
17-May
24-May
31-May
0
1
2
3
4
TAN (mg/L)
NO3-N (mg/L)
NO2-N (mg/L)
PO4-P (mg/L)
A
D
B
Figure 6. The change of TAN (A), NO3-N (B), NO2-N (C), and PO4-P (D) concentrations in the inu-
ent and euent of seaweed tanks throughout the experimental period. Black bar indicates the in-
uents and seaweed tanks, white bar indicates the euents of Ulva lactuca tanks, and grey bar indi-
cates the euents of Devaleraea mollis tanks (n = 3).
Figure 5. Correlation analysis between the growth rate of Devaleraea mollis and Ulva lactuca, and
environmental factors. Red circles indicate a positive correlation, and blue circles indicate a negative
correlation. Numbers in each cell represent the correlation coefficient. Note: M L indicates mean light
intensity; MD L indicates median light intensity.
3.4. Nutrient Concentration and Removal Efficiency
The TAN, NO
3
-N, NO
2
-N, and PO
4
-P concentrations in the influents of seaweed
tanks varied greatly among sampling dates (Figure 6). Based on the difference between
influent and effluent measurements, the nutrient removal efficiency was 37.27
±
29.25% and
44.50
±
30.70% for TAN; 56.67
±
34.34% and 65.69
±
31.81% for NO
3
-N; 29.67
±
12.61% and
22.92
±
36.67% for NO
2
-N; and 50.95
±
32.47% and 44.26
±
17.74% for PO
4
-P by D. mollis
and U. lactuca, respectively. The removal efficiency of each nutrient was not significantly
different between D. mollis and U. lactuca (p= 0.64–0.88).
Fishes 2024, 9, x FOR PEER REVIEW 8 of 16
Growth
TAN
NO3-N
NO2-N
PO4-P
N/PpH T
M L
MD L
Growth
TAN
NO3-N
NO2-N
PO4-P
N/P
pH
T
M L
MD L
0.031
−0.084−0.83
0.38 0.36−0.35
−0.54−0.60 0.70−0.061
0.49 0.73−0.560.090−0.88
0.35−0.36 0.19−0.81 0.81
−0.62 0.64−0.310.0140.14 0.13 −0.38
0.56−0.045−0.00610.34 −0.35 0.21 0.61 −0.52
0.15 0.16−0.26 0.20−0.35 0.18 0.46−0.19 0.85
0.78
−1
−0.8
−0.6
−0.4
−0.2
0
0.2
0.4
0.6
0.8
1
Devaleraea mollis
Growth
TAN
NO3-N
NO2-N
PO4-P
N/PpH T
M L
MD L
Growth
TAN
NO3-N
NO2-N
PO4-P
N/P
pH
T
M L
MD L
2.3E−4
0.36−0.76
0.067 0.33−0.40
−0.58 0.50 0.048
0.64−0.26−0.085−0.88
−0.39 0.36−0.200.067−0.84 0.83
0.51 0.64−0.28−0.00540.11 0.15−0.32
0.12−0.082−0.19 0.43−0.028−0.22 0.22−0.46
0.097 0.12−0.35 0.28 −0.14−0.0980.24−0.20 0.86 −1
−0.8
−0.6
−0.4
−0.2
0
0.2
0.4
0.6
0.8
1
Ulva lactuca
0.30
−0.60
Figure 5. Correlation analysis between the growth rate of Devaleraea mollis and Ulva lactuca, and
environmental factors. Red circles indicate a positive correlation, and blue circles indicate a negative
correlation. Numbers in each cell represent the correlation coecient. Note: M L indicates mean
light intensity; MD L indicates median light intensity.
3.4. Nutrient Concentration and Removal Eciency
The TAN, NO3-N, NO2-N, and PO4-P concentrations in the inuents of seaweed tanks
varied greatly among sampling dates (Figure 6). Based on the dierence between inuent
and euent measurements, the nutrient removal eciency was 37.27 ± 29.25% and 44.50
± 30.70% for TAN; 56.67 ± 34.34% and 65.69 ± 31.81% for NO3-N; 29.67 ± 12.61% and 22.92
± 36.67% for NO2-N; and 50.95 ± 32.47% and 44.26 ± 17.74% for PO4-P by D. mollis and U.
lactuca, respectively. The removal eciency of each nutrient was not signicantly dierent
between D. mollis and U. lactuca (p = 0.64–0.88).
0.00
0.05
0.10
0.15
0.20
0.25
0.0
0.2
0.4
0.6
0.8
1.0
29-Mar
5-Apr
12-Apr
19-Apr
26-Apr
3-May
17-May
24-May
31-May
0.000
0.002
0.004
0.006 C
29-Mar
5-Apr
12-Apr
19-Apr
26-Apr
3-May
17-May
24-May
31-May
0
1
2
3
4
TAN (mg/L)
NO3-N (mg/L)
NO2-N (mg/L)
PO4-P (mg/L)
A
D
B
Figure 6. The change of TAN (A), NO3-N (B), NO2-N (C), and PO4-P (D) concentrations in the inu-
ent and euent of seaweed tanks throughout the experimental period. Black bar indicates the in-
uents and seaweed tanks, white bar indicates the euents of Ulva lactuca tanks, and grey bar indi-
cates the euents of Devaleraea mollis tanks (n = 3).
Figure 6. The change of TAN (A), NO
3
-N (B), NO
2
-N (C), and PO
4
-P (D) concentrations in the
influent and effluent of seaweed tanks throughout the experimental period. Black bar indicates the
influents and seaweed tanks, white bar indicates the effluents of Ulva lactuca tanks, and grey bar
indicates the effluents of Devaleraea mollis tanks (n = 3).

Fishes 2024,9, 417 9 of 16
3.5. Tissue Chemical Composition and Nutrient Removal Rate
The average nitrogen content of D. mollis and U. lactuca was 4.89
±
0.058% DW
and 3.48
±
0.10% DW, and the average carbon content was 30.98
±
0.29% DW and
28.83
±
0.78% DW, respectively (Figure 7). Based on the nitrogen and carbon content
and the productivity of D. mollis and U. lactuca, the average nitrogen removal rate by U.
lactuca was 0.88
±
0.57 g/m
2
d, and by D. mollis was 0.71
±
0.46 g/m
2
d, respectively, and
the average carbon removal rate by U. lactuca and D. mollis was 7.21
±
4.54 g/m
2
d and
4.46 ±2.78 g/m2d, respectively (Table 2).
Fishes 2024, 9, x FOR PEER REVIEW 9 of 16
3.5. Tissue Chemical Composition and Nutrient Removal Rate
The average nitrogen content of D. mollis and U. lactuca was 4.89 ± 0.058% DW and
3.48 ± 0.10% DW, and the average carbon content was 30.98 ± 0.29% DW and 28.83 ± 0.78%
DW, respectively (Figure 7). Based on the nitrogen and carbon content and the productiv-
ity of D. mollis and U. lactuca, the average nitrogen removal rate by U. lactuca was 0.88 ±
0.57 g/m2d, and by D. mollis was 0.71 ± 0.46 g/m2d, respectively, and the average carbon
removal rate by U. lactuca and D. mollis was 7.21 ± 4.54 g/m2d and 4.46 ± 2.78 g/m2d, re-
spectively (Table 2).
30-Mar
6-Apr
13-Apr
20-Apr
4-May
11-May
18-May
25-May
1-Jun
7-Jun
2.8
3.2
3.6
4.0
4.4
4.8
5.2
5.6
Nitrogen (%DW)
Carbon (%DW)
30-Mar
6-Apr
13-Apr
20-Apr
4-May
18-May
25-May
1-Jun
7-Jun
22
24
26
28
30
32
34
Figure 7. The nitrogen and carbon content (% DW) of D. mollis (left) and U. lactuca (right) cultivated
in the euent of white seabass tanks. The bars represent the nitrogen content (%DW), and the line
with square symbols represents the carbon content (% DW) (n = 3).
Table 2. The average productivity (g DW/m2/d), nitrogen (N) and carbon (C) contents (% DW), and
nitrogen and carbon removal rates (g/m2/d) by Ulva lactuca and Devaleraea mollis throughout the
experimental period (n = 3).
Species
Ulva lactuca
Devaleraea mollis
Productivity (g DW/m2/d)
24.53 ± 15.34
14.40 ± 9.09
Nitrogen (N, % DW)
3.48 ± 0.10
4.89 ± 0.058
Carbon (C, % DW)
28.83 ± 0.78
30.98 ± 0.29
N removal rate (g/m2/d)
0.88 ± 0.57
0.71 ± 0.46
C removal rate (g/m2/d)
7.21 ± 4.54
4.46 ± 2.78
4. Discussion
4.1. Performance of Atractoscion Nobilis
In the present study, the calculated SGR of A. nobilis increased from an average of
0.47%/d in Phase 1 to 0.52%/d in Phase 2, while the average temperature increased from
14.25–18.61 °C in Phase 1 to 18.46–19.66 °C in Phase 2; concurrently, the calculated FCR
decreased from 2.05 in Phase 1 to 1.64 in Phase 2, which was in accordance with the results
reported in other references [19,20,33].
TAN and NO2-N are key indicators of water quality for marine nsh aquaculture
[34]. Unfortunately, no studies have been published on the toxicity of un-ionized ammo-
nia (NH3-N) and NO2-N to A. nobilis. The TAN concentrations of 0.03–0.19 mg/L in the
euents of A. nobilis tanks in the present study were similar to values reported by Draw-
bridge et al. [20] even though the stocking density of A. nobilis was increased to 30 kg/m3;
however, the NO2-N concentration of 0–0.005 mg/L was signicantly lower in the present
study compared with previous results. The reasons for those dierences are probably due
to dierences in the IMTA structures, system parameters, and experimental seasons. The
highest NH3-N concentration was estimated at 0.008 mg/L in the euents of A. nobilis
tanks based on the TAN concentration, temperature, pH, and salinity in the present study,
Figure 7. The nitrogen and carbon content (% DW) of D. mollis (left) and U. lactuca (right) cultivated
in the effluent of white seabass tanks. The bars represent the nitrogen content (%DW), and the line
with square symbols represents the carbon content (% DW) (n = 3).
Table 2. The average productivity (g DW/m
2
/d), nitrogen (N) and carbon (C) contents (% DW), and
nitrogen and carbon removal rates (g/m
2
/d) by Ulva lactuca and Devaleraea mollis throughout the
experimental period (n = 3).
Species Ulva lactuca Devaleraea mollis
Productivity (g DW/m2/d) 24.53 ±15.34 14.40 ±9.09
Nitrogen (N, % DW) 3.48 ±0.10 4.89 ±0.058
Carbon (C, % DW) 28.83 ±0.78 30.98 ±0.29
N removal rate (g/m2/d) 0.88 ±0.57 0.71 ±0.46
C removal rate (g/m2/d) 7.21 ±4.54 4.46 ±2.78
4. Discussion
4.1. Performance of Atractoscion Nobilis
In the present study, the calculated SGR of A. nobilis increased from an average of
0.47%/d in Phase 1 to 0.52%/d in Phase 2, while the average temperature increased from
14.25–18.61
◦
C in Phase 1 to 18.46–19.66
◦
C in Phase 2; concurrently, the calculated FCR
decreased from 2.05 in Phase 1 to 1.64 in Phase 2, which was in accordance with the results
reported in other references [19,20,33].
TAN and NO
2
-N are key indicators of water quality for marine finfish aquaculture [
34
].
Unfortunately, no studies have been published on the toxicity of un-ionized ammonia (NH
3
-
N) and NO
2
-N to A. nobilis. The TAN concentrations of 0.03–0.19 mg/L in the effluents of A.
nobilis tanks in the present study were similar to values reported by Drawbridge et al. [
20
]
even though the stocking density of A. nobilis was increased to 30 kg/m
3
; however, the NO
2
-
N concentration of 0–0.005 mg/L was significantly lower in the present study compared
with previous results. The reasons for those differences are probably due to differences in
the IMTA structures, system parameters, and experimental seasons. The highest NH
3
-N
concentration was estimated at 0.008 mg/L in the effluents of A. nobilis tanks based on

Fishes 2024,9, 417 10 of 16
the TAN concentration, temperature, pH, and salinity in the present study, which was
significantly lower compared with the accepted level of 0.07 mg/L in culturing juvenile
European sea bass (Dicentrarchus labrax) [
35
] and 0.06 mg/L in the culture of juvenile longfin
yellowtail (Seriola rivoliana) [
36
]. The NO
2
-N concentration in the effluents of A. nobilis was
also significantly lower than 0.21 mg/L of NO
2
-N in one RAS in which Seriola lalandi was
cultured for 488 days [
34
], and also considerably lower than the toxic levels of NO
2
-N in
other marine fish species [
37
–
40
]. Based on these results, it was indicated not only that
dissolved nutrient concentrations in the present study were safe for A. nobilis, but also
that the stocking density of A. nobilis could be increased to higher levels to produce more
nutrients for downward co-culturing seaweeds to produce more biomass and improve
their nutritional quality.
4.2. Growth and Productivity of Ulva lactuca and Devaleraea Mollis
The productivity of U. lactuca ranged from 11.31 to 47.52 g DW/m
2
d with an av-
erage of 24.53
±
15.34 g DW/m
2
d in the present study, which was within the range of
6.73–55 g DW/m
2
d reported by other researchers regardless of experimental
conditions [
6
,
20
,
22
,
41
–
45
]. However, differences in experimental conditions and the types
of culture tanks used among these studies can make comparisons difficult and the results
hard to reproduce [
6
,
20
]. In tumbled-culture tanks, U. lactuca would have higher productiv-
ity when cultivated in shallow tanks compared with relatively deeper tanks under the same
conditions because they can access sunlight for more time to process photosynthesis [
6
]. For
example, the average productivity of U. lactuca reached up to 33.83 g DW/m
2
d cultivated
in 28 cm depth tanks supplied with 0.11–0.18 mg/L TAN [
20
]. Dissolved inorganic nutrient
supplements can also influence U. lactuca productivity. When cultivated in 60 cm-depth
tumbled-culture tanks, U. lactuca achieved the maximal production of 55 g DW/m
2
d when
the inflow ammonia concentration was at 78
µ
mol/L (
≈
1.41 mg/L), and it was suggested
that the U. lactuca productivity was enhanced with the increasing of inflow ammonia
concentrations [
43
]. In the present study, U. lactuca growth did not show any correlation
with TAN concentrations mainly because of the low TAN concentrations. Under low
TAN concentration conditions, U. lactuca will uptake NO
3
-N to meet their requirements
to produce biomass, which was indicated by the positive relationship we found between
SGR and NO
3
-N concentration (Figure 5). Dissolved inorganic nitrogen was the limiting
factor for U. lactuca growth in this IMTA system because of the positive correlation with
the ratio of N/P and the negative correlation with the PO
4
-P concentrations. Ulva lactuca
productivity could likely be increased with more TAN supplements, but this process could
only be achieved through the combination effect of other environmental factors, mainly
including light level, temperature, and pH [
6
]. Conversely, U. lactuca productivity might
not be significantly improved at higher TAN concentrations if other environmental factors
are unfavorable [
6
,
22
]. In the present study, U. lactuca growth and productivity increased
significantly when seawater temperature gradually increased to what has been reported as
an optimum of 19
◦
C at the end of this trial [
46
]. Low growth rates and productivity of U.
lactuca from mid-April to the beginning of May were mainly due to low light levels [
47
].
Some researchers have indicated that low growth rates resulting from low nutrient uptake
rates could be associated with a deficiency of dissolved inorganic carbon (DIC) [
42
,
48
].
In the present study, pH remained below 9.0 throughout the experimental period, which
indicated that there was no DIC limitation for U. lactuca growth [
49
]. However, the negative
correlation between Ulva growth and pH suggests that supplying more DIC might increase
the growth and productivity of U. lactuca, which could be achieved by increasing the initial
stocking densities of co-cultured A. nobilis, or aerating pure CO
2
into this IMTA system,
especially during the daytime. Also, in this IMTA system, extra A. nobilis or invertebrates
such as abalone, sea cucumbers, sea urchins, and filter feeders (e.g., oysters, mussels)
can be cultured in second-tier tanks to produce additional TAN and DIC for U. lactuca to
further enhance productivity. In addition to temperature, light level, nutrient type, and
concentration, seawater exchange rate would be an important factor impacting the growth

Fishes 2024,9, 417 11 of 16
and productivity of U. lactuca in tumbled culture tanks in IMTA systems. The seawater ex-
change rate in U. lactuca tanks was set to 63 vol./day in this study, which was demonstrated
as the optimum (compared with 4 and 12 vol./day) by Drawbridge et al. [
20
]. Therefore,
the effects of seawater exchange rates higher than 63 vol./day and values between 12 to
63 vol./day on the growth, productivity, and nutritional quality of U. lactuca should be
tested in future studies.
The growth, productivity, and nutritional composition of D. mollis have already been
studied by some researchers for the purpose of optimizing cultivation conditions, including
nutrient concentrations, stocking density, light, salinity, temperature, seawater exchange
rate, pH, aeration rate, and cultivation method [
25
,
28
,
30
,
31
,
50
–
57
]. Gadberry et al. [
27
]
also cultivated D. mollis for one year in land-based tanks with effluents from a fish-rearing
system, and the fish effluents were pulse-fertilized twice a week with 5.00 mg/L calcium
nitrate as nitrogen, to evaluate seasonal growth, yield, nutritional composition, and con-
taminant levels. It is not practical to directly compare our results with these studies due
to differences in experimental conditions and types of culture tanks. Devaleraea mollis is a
temperate species and in general, grows well when seawater temperatures are lower than
16
◦
C. The optimal temperatures for maximum SGR were found to be a function of light,
with increased light supporting higher growth rates at a higher temperature due to an
interaction between light and temperature [
31
]. In the study by Demetropoulos and
Langdon [
31
], D. mollis grew well at 18
◦
C at high specific light density (SLD, 0.021 mol pho-
tons/g/day). During the present study, the average temperature reached over 18 ◦C after
April 25
th
, and the mean SLD maintained high levels (0.015–0.036 mol photons/g/day with
an average of 0.020 mol photons/g/day) from the beginning of May except on three over-
cast days from May 26 to 28 (Figure 2). This can partially explain why D. mollis maintained a
relatively high productivity at 5.98–21.50 g DW/m
2
d (except for 1.23 g DW/m
2
d evaluated
on June 1 due to overcast days during that period) even though the seawater temperature
went over 18
◦
C. Nutrient types (TAN, or NO
3
-N and PO
4
-P), as well as their concentra-
tions, are other factors that will impact the growth and productivity of D. mollis, especially
during high-temperature periods (e.g., >18
◦
C). Demetropoulos and Langdon [
30
] recom-
mended providing high nitrogen (NO
3
-N) fertilization rates of
2353–2942 µmol/d
in order
to maintain high growth rates of D. mollis at 16
◦
C and light levels of 300–1400
µ
mol/m
2
/s.
It is hard to apply this approach in a flow-through aquaculture system because the seawater
exchange rate is another important factor that will significantly impact the growth and pro-
ductivity of D. mollis in tumbled cultivation tanks. It was previously shown that 70–100% of
the nitrogen requirement of macroalgae was provided by co-cultured fish [
27
], based on an
influent ammonia–nitrogen concentration of 20–30
µ
mol/L (0.36–0.54 mg/L) [
58
]. It could
be concluded that the TAN concentration (0.02–0.19 mg/L) was not sufficient for D. mollis to
maintain a high growth rate in the present study, which was also indicated by the positive
correlation between the growth rate and N/P ratio. The appropriate molar N/P ratio for
nutrient supplementation was suggested to be approximately 70 and 35 for maximum
D. mollis growth under low and high light conditions, respectively [
30
]. The molar N/P
ratio of 0.60 to 6.11 in the influents of D. mollis culture tanks also indicated that dissolved
inorganic nitrogen was the limiting factor for D. mollis to achieve high productivity in the
present study. The negative correlation between the growth of D. mollis and pH suggests
that supplying more DIC might increase the growth and productivity of D. mollis, even
though DIC limitation was not indicated by pH values (<9.0). As mentioned above, the
growth rate of D. mollis could also be significantly impacted by seawater exchange rates.
The growth, productivity, and nutritional quality of D. mollis were shown to be significantly
higher when cultivated at an exchange rate of 35 vol./day compared with those of 1 and
6 vol./day
[
54
,
55
]. Demetropoulos and Langdon [
31
] stated that the high yield and growth
of D. mollis were achieved at the seawater exchange rate of 60 vol./day in combination with
moderate stocking density and high natural light. In the present study, the productivity
and nutritional quality of D. mollis were only tested at 63 vol./day of seawater exchange.

Fishes 2024,9, 417 12 of 16
Therefore, studies to determine differences in the growth, productivity, and nutritional
quality of D. mollis across a wide range of seawater exchange rates should be conducted.
Seaweed culture can be successful in relatively deep tanks when properly “tumbled”
in the water column using aeration to bring suspended seaweeds from deeper layers to
the surface to access light for photosynthesis [
20
]. Strong aeration evens out light exposure
and facilitates solute diffusion [
59
]. The turbulence generated by the aeration also thins
the diffusive boundary layer (DBL) around the frond surfaces, accelerating the inflow of
nutrients to the fronds and removal of excess oxygen from them [
42
], which ultimately
enhances the nutrient uptake rate, growth, and productivity of seaweeds in tumble culture
tanks [
25
,
60
]. In this present study, the growth of U. lactuca and D. mollis was only evaluated
at one aeration rate of 25 L/min in 700 L tanks (0.035 L air/L of seawater/min), but it is
expected that the performance of these two species would be impacted by the interaction
between nutrient concentration, seawater exchange rate, and aeration rate in tumble culture
tanks. These interactions are worthy to be evaluated in future studies.
4.3. Water Quality Remediation and Nutrient Uptake Rates
Co-cultured seaweeds can significantly increase pH values in the effluent from primary-
fed species by utilizing DIC during the photosynthesis process. In one study conducted by
Huo et al. [
6
], seawater pH in U. lactuca tanks gradually increased from 7.84 to a peak of
8.59 during the daytime, and gradually decreased to a low of 7.76 before dawn the next
day when integrated into an IMTA system co-culturing with S. dorsalis. In an IMTA system
integrating red abalone (Haliotis rufescens) and D. mollis, the mean seawater pH increased
by 0.2 pH units due to the biological activity of D. mollis [
13
]. In the present study, U. lactuca
and D. mollis increased pH by 0.01–0.73 units in the seawater effluents compared to the
influents. The pHs of the effluents from A. nobilis tanks were lower from mid-May to the
end of the trial because the feeding rate of A. nobilis increased from 1.07 to 1.42% body
weight per day when water temperatures increased (Table 1). During this time, the pH
values only increased 0.01–0.14 pH units higher in the effluents from the seaweed tanks
(Figure 3). This was mainly caused by the reduced growth and productivity of D. mollis at
higher water temperatures. As discussed above, increasing light levels and/or nutrient
concentrations, optimizing seawater exchange rates and/or aeration rates, might increase
the growth and productivity of D. mollis, but because of its intrinsic properties, the growth
of D. mollis would continue to decrease with increasing temperature. During seasons when
water temperature is >18
◦
C, some other economically valuable, and temperature-tolerant
red seaweeds, such as Gracilaria parvispora [
61
] or Gracilaria pacifica [
62
], could be integrated
into the IMTA system to replace D. mollis.
Nitrogen removal rates are dependent on the productivity and tissue nitrogen concen-
tration of seaweeds cultivated in the effluents from primary-fed species in IMTA systems.
The average productivity was 24.53
±
15.34 g DW/m
2
d and 14.40
±
9.09 g DW/m
2
d for
U. lactuca and D. mollis during the present study, which could be enhanced by optimizing
the cultivation parameters discussed above. The tissue nitrogen concentration of seaweeds
functions as dissolved inorganic nitrogen load levels within a certain range under suit-
able conditions of other parameters in tumbled culture tanks. In the present study, the
average nitrogen content of U. lactuca (3.48% DW) was similar to another study in which
fish effluent TAN concentrations were similar [
20
]. However, the nitrogen content in the
present study was significantly lower compared with that of Huo et al. [
6
] where Ulva
nitrogen content reached 4.85% DW under TAN concentrations that reached as high as
1.19 mg/L. Similarly, nitrogen content was lower compared with 4.41–7.27% DW when
TAN in effluent from a fishpond ranged 1.18–2.23 mg/L [
22
]. Neori et al. [
43
] conducted a
series of trials to demonstrate that the nitrogen content of U. lactuca improved significantly
when TAN concentration increased from 10
µ
M to 48
µ
M in effluents from fishponds.
Similar to findings for U. lactuca, the nitrogen content of D. mollis was shown to be a
function of nitrate (NO
3
-N) concentrations and averaged between 3.07 and 5.01% DW [
52
],
which indicates that the protein content of D. mollis will improve with increasing dissolved

Fishes 2024,9, 417 13 of 16
inorganic nitrogen. The average nitrogen content of D. mollis (4.89% DW) in this study was
higher than most values (3.07–5.01% DW) reported by Demetropoulos and Langdon [
52
].
It was also higher than all values (3.76–4.11% DW) across four seasons by [
27
] when D.
mollis was cultivated in the effluent of fish tanks. It was also reported that the nitrogen
content of D. mollis was a function of nutrient application rate [
30
]. Specifically, when D.
mollis was cultivated at nutrient supplementation intervals of daily and every 3 and 5 days,
the nitrogen content of 4.99–5.83% DW was higher than other nutrient supplementation
intervals and also higher than the values obtained in the present study. This demonstrated
that continuously supplying high concentrations of dissolved inorganic nitrogen to D.
mollis can significantly increase tissue nitrogen contents, which will ultimately increase the
protein content of D. mollis and promote higher nutrient removal rates. In the present study,
TAN and NO
3
-N concentrations in effluents from A. nobilis tanks varied greatly between
experimental days with relatively low levels on some days, which slowed down the growth
and productivity of D. mollis and decreased the tissue nitrogen content. Based on this
discussion, we concluded that the tissue nitrogen content of U. lactuca and D. mollis could
be further enhanced by supplying higher concentrations of nutrients through increased
initial stocking density of A. nobilis and culturing invertebrates in second-tier tanks to
increase nutrient supplementation.
5. Conclusions
Atractoscion nobilis is a marine fish species that is ready to be cultured commercially
in the United States, but little research has been done to study the integration of A. nobilis
with low trophic level organisms. The present study reported the growth, productivity,
nutritional quality, and nutrient removal rate of economically valuable seaweeds U. lac-
tuca and D. mollis when integrated with A. nobilis in a land-based flow-through cascade
IMTA system. U. lactuca and D. mollis achieved high growth rates and productivities,
nutritional qualities, and nutrient removal rates. Due to its temperature constraints, D.
mollis can be integrated into IMTA systems during late fall and early summer (November
to June) in southern California. The performance of D. mollis can be enhanced by increasing
light levels (reducing shade) during overcast days and during high-temperature periods.
The performance of U. lactuca and D. mollis can both be enhanced through consistently
supplying high concentrations of nutrients in influents. The effects of aeration rates and
their interactive effects with seawater exchange rates on the performance of U. lactuca
and D. mollis should be investigated to further maximize the nutrient removal rates and
diversify the seafood production in IMTA systems in future studies. Moreover, nitrogen
and phosphorus concentrations in the environment of A. nobilis tanks would be signifi-
cantly reduced by the assimilation of U. lactuca and D. mollis, which will be qualified in
future studies.
Author Contributions: Y.H., M.S.E. and M.D. designed the experiment. M.S.E. and Y.H. conducted
the experiment and data analysis. Y.H. drafted the manuscript. Y.H., M.S.E. and M.D. revised the
manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Builders Initiative (BIF Grant 2022-6439).
Institutional Review Board Statement: All experiments were conducted according to the Institutional
Animal Care and Use Committee (IACUC) of HSWRI on the care and use of experimental animals.
Animal research in this study gained approval from the IACUC (identification code: 2022-05).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article. The data that support the findings
of this study are available from the corresponding author upon reasonable request.
Acknowledgments: This study received additional support from California’s Ocean Resources
Enhancement and Hatchery Program. The authors would like to thank Matthew Edward’s laboratory
at San Diego State University for measuring the nutritional composition of the seaweeds. Many
technicians supported the studies reported here; we are thankful for their contributions.

Fishes 2024,9, 417 14 of 16
Conflicts of Interest: The authors declare no conflicts of interest.
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