fishes

Article
Experimental Study of the Environmental Effects of
Summertime Cocultures of Seaweed Gracilaria lemaneiformis
(Rhodophyta) and Japanese Scallop Patinopecten yessoensis in
Sanggou Bay, China
Yi Liu 1 , Xinmeng Wang 1 , Wenguang Wu 1 , Jun Yang 1 , Ningning Wu 2 and Jihong Zhang 1,3, *

1 Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea
Fisheries Research Institute, Chinese Academy of Fishery Science, Qingdao 266071, China;
[email protected] (Y.L.); [email protected] (X.W.); [email protected] (W.W.); [email protected] (J.Y.)
2 Division of Aquatic Products Quality and Safety, Qingdao Municipal Marine Development Bureau,
Qingdao 266071, China; [email protected]
3 Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National
Laboratory for Marine Science and Technology, Qingdao 266000, China
* Correspondence: [email protected]

Abstract: The shellfish–algae mode of integrated multitrophic aquaculture (IMTA) is a sustainable



aquaculture method that benefits the environment and the carbon cycle. However, most current


shellfish–algae aquaculture modes are based on the expansion of kelp aquaculture. Due to the low
Citation: Liu, Y.; Wang, X.; Wu, W.; tolerance of kelp to high temperatures, integrated shellfish–algae aquaculture areas often become
Yang, J.; Wu, N.; Zhang, J. shellfish monocultures in summer, which may lead to both high mortality rate of shellfish and to
Experimental Study of the economic loss while causing serious environmental harm via eutrophication, decreases in dissolved
Environmental Effects of oxygen (DO), and decreases in pH. In this study, we investigated the effects of different ratios
Summertime Cocultures of Seaweed
of seaweed (Gracilaria lemaneiformis), which is tolerant of high temperatures, to Japanese scallop
Gracilaria lemaneiformis (Rhodophyta)
(Patinopecten yessoensis) on water quality and environmental parameters. A two-day small-scale
and Japanese Scallop Patinopecten
enclosure water body experiment was conducted in Sanggou Bay (Shandong, China) in August 2019.
yessoensis in Sanggou Bay, China.
The results demonstrated that culturing shellfish alone significantly affected pH, DO, eutrophication,
Fishes 2021, 6, 53. https://doi.org/
10.3390/fishes6040053
and other environmental indicators, as well as the carbonate system. The negative environmental
impact of the shellfish–algae aquaculture system was much smaller. However, too high a proportion
Academic Editor: of algae might consume excessive amounts of dissolved inorganic nitrogen (DIN) and nutrients,
Bernardo Baldisserotto while too low a proportion of algae might not fully absorb the nutrients released by the cultured
shellfish, in turn leading to an increased risk of eutrophication. The shellfish–algae aquaculture
Received: 25 August 2021 system not only improved the inorganic carbon system, but also the organic carbon system. At the
Accepted: 19 October 2021 end of the experiment, all the parameters of the inorganic carbon system had decreased significantly,
Published: 22 October 2021 while all the parameters of the organic carbon system had increased significantly. The results of this
study illustrate the need to include macroalgae rotations in summer, and that an appropriate ratio of
Publisher’s Note: MDPI stays neutral
shellfish to algae is necessary to achieve a sustainable aquaculture system. Moreover, this research
with regard to jurisdictional claims in
has also confirmed the importance of the future and related research in the actual production, which
published maps and institutional affil-
will provide useful information to guide governmental strategies for summer aquaculture rotations
iations.
and insight into the controversy concerning whether aquaculture is a carbon source or sink.

Keywords: integrated multitrophic aquaculture (IMTA); Gracilaria lemaneiformis; Patinopecten

yessoensis; Sanggou Bay
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
1. Introduction
conditions of the Creative Commons
Attribution (CC BY) license (https:// The development of aquaculture is essential to meet growing human food require-
creativecommons.org/licenses/by/ ments. However, aquaculture can also significantly enrich waters with organic matter and
4.0/). nutrients [1], thus making them more susceptible to eutrophication and which restricts

Fishes 2021, 6, 53. https://doi.org/10.3390/fishes6040053 https://www.mdpi.com/journal/fishes

Fishes 2021, 6, 53 2 of 16

the broadscale application of aquaculture techniques. In order to overcome these prob-

lems, there is a growing global interest in the integrated multitrophic aquaculture (IMTA)
mode [2]. IMTA cocultivates species of different trophic levels and ecological niches with
complementary functions to achieve a healthy and sustainable aquaculture system [3]. The
shellfish–algae IMTA mode has proven to be the only economical solution for recycling
wasted nutrients in open water [2,4]. Additionally, cultured shellfish and algae play an
important role in the coastal biocarbon cycle [5]. Carbon can be removed from the coastal
ecosystem through the harvesting of shellfish and algae [6], thereby increasing the carbon
sequestration capacity of marginal shelf seas, accelerating the absorption of atmospheric
CO2 by the ocean, and alleviating the global greenhouse effect. Therefore, shellfish–algae
farming is an efficient and economically important mode of environmentally friendly
aquaculture that benefits the environment and the coastal carbon cycle [6,7]. However,
most studies on the role of shellfish–algae culture in the carbon cycle have focused on the
inorganic carbon cycle, and few have considered its impact on organic carbon [7,8].
At present, the most successful shellfish–algae IMTA modes have been based on the
expansion of kelp culture [9]. However, due to the intolerance of kelp to high temperatures,
many shellfish–algae IMTA areas in northern China have large areas where only shellfish
are being cultured for up to four months during summer. For example, Sanggou Bay is a
key shellfish–algae IMTA farming base in North China that produces about 80,000 tons
of kelp annually [10]. After the summer kelp is harvested, about 1/6 of the kelp farming
area will be used to cultivate another macroalgae species, namely the seasonal rotational
aquaculture of macroalgae. The annual production of these rotated algae accounts for
approximately 1/8 of the kelp yield (data from local government). The physiological activi-
ties of shellfish without active algae may stress the natural ecosystems of the aquaculture
areas [11]. Additionally, the stratification of seawater caused by freshwater input and high
temperatures make eutrophication more likely in summer [12,13]. The combination of
multiple environmental stressors such as temperature and eutrophication may also lead to
a higher incidence of aquatic diseases in summer [14–16]. Japanese scallop (Patinopecten
yessoensis) is a scallop that has become a major aquaculture species in northern China,
covering the coastal area from Liaoning Province to Shandong Province. Recently, massive
die-offs of Japanese scallop have been observed in Sanggou Bay during summer. This may
be attributed to both extreme high temperatures and the absence of macroalgal cultures
in summer [2,17].
Seaweed (Gracilaria lemaneiformis) is an algae with a high commercial value and a
strong bioremediation effect [17–19] which grows fast, has a short growth cycle, and
can be harvested in 2–3 months. This species can also adapt well to high temperature
environments, so a rotation strategy that includes seaweed in summer can bring significant
economic and environmental benefits. The physiological activity of this species in summer
may also improve the conditions for the shellfish and reduce the frequency of summer
shellfish die-offs. Therefore, in this study the effects of different Japanese scallop/seaweed
culture modes on the marine environment and the local ecosystem were investigated in
Sanggou Bay during summer. The objectives of this study were to identify the different
environmental and ecological impacts of different culture modes, and to explore the
benefits and feasibility of rotations that include seaweed in summer. We evaluated the
impacts of shellfish–algae IMTA cultures on the marine environment and carbon cycle,
identified any biologically mutual benefits, and illustrated the advantages of the IMTA
culture mode. These results can be used as a reference for sustainable aquaculture activities
in different regions.

2. Materials and Methods

2.1. Experiment Design and Management
A relatively closed ecosystem was constructed with water that simulated the water
quality in Sanggou Bay (see Figure 1). Seaweed and Japanese scallop (wet weight ~9.5 g,
shell height ~5 cm) obtained from Shandong Rongcheng Xunshan Group were used as
 Fishes 2021, 6, x FOR PEER REVIEW 3 of 16

Fishes 2021, 6, 53 2. Materials and Methods 3 of 16

2.1. Experiment Design and Management
A relatively closed ecosystem was constructed with water that simulated the water
quality in Sanggou Bay (see Figure 1). Seaweed and Japanese scallop (wet weight ~9.5 g,
the
shellIMTA
heightculture components
~5 cm) obtained from in this study,
Shandong and theXunshan
Rongcheng experiments
Groupwerewerecarried out using a
used as the
multifunctional experimental
IMTA culture components enclosure
in this study, and device patented bywere
the experiments the carried
State Intellectual
out using aProperty
Office of China,experimental
multifunctional as shown in Figure 2.
enclosure The patented
device enclosure bybag had aIntellectual
the State diameterProperty
of 1 m, a height
Office
of 2 m,ofand
China, as shownofinabout
a volume Figure1.5 m3 enclosure
2. The filled with bag had a diameter
seawater. of 1 m, a height
The Japanese scallops were
of 2 m, and
scrubbed a volume
with a brushof and
aboutplaced
1.5 m3on filled with seawater.
a plastic tray andThe the Japanese
seaweedscallops were
were suspended by
scrubbed with a brush and placed on a plastic tray and the seaweed were
a rope, then both were put into the enclosure bag which was filled with seawater and suspended by a
rope, then
sealed by both were put
expelling all into
the the
air.enclosure bag which
The enclosures wasfixed
were filled with seawater raft
to a floating and sealed
on the coastal
by expelling all the air. The enclosures were fixed to a floating raft on the coastal sea
sea surface. Four experimental groups and one blank control group were established, with
surface. Four experimental groups and one blank control group were established, with
three replicates in each group. The experiment was conducted from 18:00 on August 11 to
three replicates in each group. The experiment was conducted from 18:00 on August 11 to
18:00 on August 13, 2019 (48 h). The biomasses of the different experimental groups are
18:00 on August 13, 2019 (48 h). The biomasses of the different experimental groups are
shown
shown in inTable
Table1.1.Sampling
Sampling waswas conducted
conducted at 12ath 12 h intervals.
intervals. The sampling
The sampling times
times were set were set
to coincide with the local sunrise and sunset
to coincide with the local sunrise and sunset times. times.

Fishes 2021, 6, x FOR PEER REVIEW 4 of 16

Figure 1. The study site.
Figure 1. The study site.

Table 1. Density of Japanese scallop and seaweed in the different treatments.

Mode Japanese Scallop (g) Seaweed (g) Ratios

Control (0#) 0 0 /
Group 1 (1#) 400.3 ± 2.4 0 /
Group 2 (2#) 0 399.7 ± 3.22 /
Group 3 (3#) 401.3 ± 2.59 400.4 ± 7.52 1:1
Group 4 (4#) 398.87 ± 8.98 639.08 ± 6.15 1:1.6

Figure
Figure 2.2. Experimental
Experimental device: 1. Plexiglas
device: barrel;
1. Plexiglas 2. experimental
barrel; bag–polyethylene
2. experimental material; 3.
bag–polyethylene material; 3.
sinker
sinker stone; 4. air bag; 5. inflatable valve; 6. tie belt; 7. buckle ring–a; 8. sealing cap; 9.ring–
stone; 4. air bag; 5. inflatable valve; 6. tie belt; 7. buckle ring–a; 8. sealing cap; 9. buckle buckle ring–b;
b; 10–11. locking buckle; 12. device for drainage; 13. rubber plugs; 14. rubber pads.
10–11. locking buckle; 12. device for drainage; 13. rubber plugs; 14. rubber pads.
2.2. Sample Analysis
The dissolved oxygen (DO), salinity (S), pH, and temperature (T) were determined
in situ using a water analyzer (YSI, Professional Plus, Yellow Spring, OH, USA). An
amount of 1000 mL water samples were collected from each replicate at each sampling
time, and filtered with a filter (Nalgene, Rochester, NY, USA). A total of 200 mL of each
Fishes 2021, 6, 53 4 of 16

Table 1. Density of Japanese scallop and seaweed in the different treatments.

Mode Japanese Scallop (g) Seaweed (g) Ratios

Control (0#) 0 0 /
Group 1 (1#) 400.3 ± 2.4 0 /
Group 2 (2#) 0 399.7 ± 3.22 /
Group 3 (3#) 401.3 ± 2.59 400.4 ± 7.52 1:1
Group 4 (4#) 398.87 ± 8.98 639.08 ± 6.15 1:1.6

2.2. Sample Analysis

The dissolved oxygen (DO), salinity (S), pH, and temperature (T) were determined in
situ using a water analyzer (YSI, Professional Plus, Yellow Spring, OH, USA). An amount
of 1000 mL water samples were collected from each replicate at each sampling time, and
filtered with a filter (Nalgene, Rochester, NY, USA). A total of 200 mL of each water sample
was filtered through a Whatman GF/F filter membrane precombusted at 450 ◦ C for 5 h. The
filtrate was passed through a total organic carbon analyzer to determine dissolved organic
carbon (DOC) and dissolved inorganic carbon (DIC), and the filter membrane was acidified
and analyzed using an elemental analyzer (Elementar EL, Langenselbold, Germany) to
measure the particulate organic carbon (POC) in the water. The partial pressure of carbon
dioxide (pCO2 ) and the components of the carbonate system were calculated using the
CO2 SYS software, and the total alkalinity (TA) required for the calculation was measured
after filtering with Whatman GF/F membranes using an 848 Titrino plus automatic titrator
(Metrohm AG, Herisau, Switzerland). A total of 500 mL of each water sample was filtered
with acetate membranes of three aperture sizes (20, 2, and 0.45 µm) sequentially. The filter
membranes were used to measure chlorophyll-a (Chl-a) contained via fluorometric analysis
by particles of different sizes, and the sum of the three groups of Chl-a was taken as the
total Chl-a concentration. The filtrate was used for the assay of nutrients, which were
assayed according to the chemical methods specified in the Chinese National Standards.

2.3. Data Analysis

Data were analyzed using SPSS 19.0 statistical software. Two factor repeated ANOVA
was used to detect the interactions between different experimental treatments and exper-
imental times for each parameter. One-way ANOVA was used to detect the differences
between different experimental times within the same experimental group, where p < 0.05
indicated a significant difference.

3. Results
None of the shellfish or algae died during the experiment. The water temperature and
salinity varied within relatively small ranges, from 22.27 ◦ C to 24.51 ◦ C and 31.8 to 32.0,
respectively, in all experimental groups.

3.1. Changes of pH, DO, and TA in Different Culture Modes

When the shellfish were cultured alone it resulted in acidification of the water and a
decrease in both DO and TA (Figure 3a–c). The effect of the experimental treatment group
and time on pH, DO, and TA were significant, and their interaction was also significant
(p < 0.05; Table 2). By the end of the experiment the pH, DO, and TA values had decreased
in group 1, which had shellfish only, but had significantly increased in the other three
experimental groups (p < 0.05). The DO showed significant diurnal variation and was
significantly higher during the day (p < 0.05, one-way ANOVA).
 When the shellfish were cultured alone it resulted in acidification of the water and a
decrease in both DO and TA (Figure 3a–c). The effect of the experimental treatment group
and time on pH, DO, and TA were significant, and their interaction was also significant
(p < 0.05; Table 2). By the end of the experiment the pH, DO, and TA values had decreased
in group 1, which had shellfish only, but had significantly increased in the other three
Fishes 2021, 6, 53 experimental groups (p < 0.05). The DO showed significant diurnal variation and was 5 of 16
significantly higher during the day (p < 0.05, one-way ANOVA).

8.8
a
8.6 a a a
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D1 18:00 D2 6:00 D2 18:00 D3 6:00 D3 18:00
Time

(a)
15
b a
a
a
c b
10 a b 0#
bc a d c cd b b cc
DO (mg/L)

c dcd
b
c 1#
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Fishes 2021, 6, x FOR PEER REVIEW 6 of 16
Time

(b)
2500 a
a b
b
2450 a b
ab c c c a a
baccc
TA (μmol/kg)

badcc 0#
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2400
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2300
D1 18:00 D2 6:00 D2 18:00 D3 6:00 D3 18:00
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(c)
Figure 3. (a)3.The
Figure (a)pH
TheofpH
modes at different
of modes times. The
at different different
times. letters indicate
The different lettersa indicate
significant difference difference
a significant
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times. The different letters indicate a significant difference of DO within mode at different detecting
times. The different letters indicate a significant difference of DO within mode at different detecting
times. Means ± SE (n = 3). (c) The TA of modes at different times. The different letters indicate a
significant Means ± of
times. difference SETA(n within
= 3). (c) TheatTA
mode of modes
different at different
detecting times.±The
times. Means SE (ndifferent
= 3). letters indicate a
significant difference of TA within mode at different detecting times. Means ± SE (n = 3).
Table 2. Two-way repeated ANOVA results of DO, pH, TA in the seawater of modes at different sampling times.

Source Sum of Squares df Mean Squares F p

Mode 239.505 4 59.876 1373.056 0.000
DO Time 120.056 4 30.014 688.265 0.000
Mode×Time 228.045 16 14.253 326.839 0.000
Mode 1.266 4 0.316 222.854 0.000
Fishes 2021, 6, 53 6 of 16

Table 2. Two-way repeated ANOVA results of DO, pH, TA in the seawater of modes at different sampling times.

Source Sum of Squares df Mean Squares F p

Mode 239.505 4 59.876 1373.056 0.000
DO Time 120.056 4 30.014 688.265 0.000
Mode × Time 228.045 16 14.253 326.839 0.000
Mode 1.266 4 0.316 222.854 0.000
pH Time 0.409 4 0.102 72.051 0.000
Mode × Time 0.672 16 0.042 29.575 0.000
Mode 36,383.561 4 9095.890 7517.265 0.000
TA Time 4551.065 4 1137.766 940.303 0.000
Mode × Time 45,434.731 16 2839.671 2346.835 0.000

3.2. Variation of Chl-a and Components in Different Culture Modes

The concentration of Chl-a showed an overall increasing trend, with the smallest
increase being in the shellfish only culture group. According to the two factor ANOVA,
total Chl-a was significantly affected by the treatment and time, as well as the interaction
between the two factors (p < 0.05; Table 3). The total Chl-a of all groups showed initial
increasing trends and then decreasing trends (Figure 4a), and the total Chl-a of group 1 at
the end of the experiment was not significantly different from that at the start (p > 0.05).
The total Chl-a of the other groups were all significantly higher at the end than that at the
beginning of the experiment (p < 0.05).

Table 3. Two-way repeated ANOVA results of Chl-a in the seawater of modes at different sampling times.

Source Sum of Squares df Mean Squares F p

Mode 21.224 4 5.306 124.961 0.000
Chl-a Time 249.662 4 62.415 1469.937 0.000
Mode × Time 30.099 16 1.881 44.303 0.000

The structural compositions of Chl-a throughout the experiment for each group are
shown in Figure 4b. The composition of Chl-a in the enclosure system was categorized by
the particle size. At the beginning of the experiment the contribution of nanophytoplankton
(2–20 µm) was the largest, accounting for 86.66% of the Chl-a, while microphytoplankton
(>20 µm) and picophytoplankton (0.45–2 µm) contributed 4.95% and 8.39%, respectively.
At the end of the experiment, the contribution of nanophytoplankton to Chl-a decreased to
66.28% and 58.25% in the control and experimental group 1, respectively, but increased to
93.68% in group 2. The contributions of Chl-a by different particle sizes in experimental
group 3 and group 4 did not significantly change during the experiment (p > 0.05).

3.3. Changes in DIC, pCO2 , and the Carbonate System in Different Aquaculture Modes
There were significant differences in DIC, pCO2 , and the carbonate system (HCO3 − ,
CO3 2− , and CO2 ) between groups and times, and the interaction between the two factors
was also significant (p < 0.05; Table 4). The DIC showed the same trend in all groups, with
significant decreases during the day and increases at night (p < 0.05, one-way ANOVA).
The DIC was significantly lower at the end of the experiment than at the beginning in
all groups (p < 0.05; Figure 5a). Among the carbonate system components, HCO3 − and
CO3 2− were significantly lower (p < 0.05) at the end of the experiment than that at the
beginning and exhibited diurnal fluctuations (Figure 5c). The changes in CO2 and pCO2
were consistent with each other. The diurnal variation of CO2 and pCO2 was obvious in
group 1, lower during the day and higher at night (p < 0.05), and their concentrations were
significantly higher at the end of the experiment than at the beginning. The CO2 and pCO2
of the other groups were significantly lower after the end of the experiment than at the
beginning of the experiment (p < 0.05). The trends in CO2 and pCO2 in groups 3 and 4
Fishes 2021, 6, 53 7 of 16

were the same and they exhibited a significant diurnal pattern (p < 0.05). At the end of the
experiment, the CO2 and pCO2 in group 4 were significantly lower than in group 3 but not
significantly different from those in group 2 (Figure 5b,c).

Table 4. Two-way repeated ANOVA results of DIC, pCO2 , and carbonate system in the seawater of modes at different
sampling times.

Source Sum of Squares df Mean Squares F p

Mode 215,895.095 4 53,973.774 10,709.048 0.000
DIC Time 7,725,702.666 4 1,931,425.667 383,218.162 0.000
Mode × Time 198,111.298 16 12,381.956 2456.730 0.000
Mode 1,664,176.577 4 416,044.144 80,420.904 0.000
pCO2 Time 307,442.537 4 76,860.634 14,857.081 0.000
Mode × Time 823,088.365 16 51,443.023 9943.883 0.000
Mode 250,760.705 4 62,690.176 2072.403 0.000
HCO3 − Time 6,416,415.953 4 1,604,103.988 53,028.231 0.000
Mode × Time 190,286.215 16 11,892.888 393.153 0.000
Mode 5749.88 4 1437.475 47.520 0.000
CO3 2− Time 66,728.177 4 16,682.044 551.473 0.000
Mode × Time 11,856.723 16 741.045 24.497 0.000
Mode 1929.804 4 482.451 15.949 0.000
CO2 Time 433.633 4 108.408 3.584 0.012
Mode × Time 1073.704 16 67.107 2.218 0.016

3.4. POC and DOC in Different Culture Modes

The POC and the DOC were significantly dependent on the treatment and time, whose
interaction was also significant (p < 0.05; Table 5). At the end of the experiment, the POC of
group 1 was significantly lower than before the experiment, while the POC of the remaining
groups were all significantly higher (p < 0.05; Figure 6a). The change of the DOC was
similar to that of the POC. After the experiment, the DOC of all groups was significantly
higher than that at the beginning (p < 0.05), except for group 1. Furthermore, the DOC of
different groups was significantly different (p < 0.05; Figure 6b).

Table 5. Two-way repeated ANOVA results of POC and DOC in the seawater of modes at different sampling times.

Source Sum of Squares df Mean Squares F p

Mode 48,740.061 4 12,185.015 50.286 0.000
DOC Time 125,626.612 4 31,406.653 129.611 0.000
Mode × Time 76,374.501 16 4773.406 19.699 0.000
Mode 1.599 4 0.400 53.790 0.000
POC Time 1.057 4 0.264 35.548 0.000
Mode × Time 1.219 16 0.076 10.248 0.000
Fishes 2021,
Fishes 6, 6,
2021, 53x FOR PEER REVIEW 7 of 16 8 of 16

16

14 a

12 a a a
b
10 c bb b 0#
b bc
Chl a (μg/L)
b
c c
8 ac a 1#
a d
6 b 2#
ebd cd
3#
4
4#
2

0
D1 18:00 D2 6:00 D2 18:00 D3 6:00 D3 18:00
Time

(a)

(b)
Figure 4. (a) The Chl-a of modes at different times. The different letters indicate a significant
difference of pH within mode at different detecting times. Means ± SE (n = 3). (b) The size fraction
of Chl-a of modes at different times.
Fishes 2021, 6, 53 9 of 16

3.5. Variation of Nutrients in Different Culture Modes

The nutrient concentrations and structural changes are shown in Table 6. At the end of
the experiment, the concentration of dissolved inorganic nitrogen (DIN) was significantly
higher in group 1, which had shellfish only, than in the other experimental groups, and
it had also significantly increased over the course of the experiment (p < 0.05). Group 1
also had significantly higher phosphate (PO4 ) than the other groups at the end of the
experiment (p < 0.05), but it was not significantly different from the beginning of the
experiment (p > 0.05). The concentration of silicate (SiO4 ) had also significantly decreased
in all experimental groups containing shellfish. The nutrients in the other groups all
Fishes 2021, 6, x FOR PEER REVIEW showed decreasing trends, with DIN and phosphate decreasing the most9 in of 17
the group 2,
which had algae only.

2500 a aa a
b b b bb a

2000 d c c
d d dd c cc e
0#
DIC (μmol/L)

e
1500 ee e
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D1 18:00 D2 6:00 D2 18:00 D3 6:00 D3 18:00
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(b)

Figure 5. Cont.
 Fishes 2021, 6, x FOR PEER REVIEW 10 of 17
Fishes 2021, 6, 53 10 of 16

2500 ci
a a
2000 bbab aa
c
bc

HCO3-(μmol/L)
d c e
1500 dcd
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250 cii
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a
200
aaaab a aa
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150 b b b
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25 c 0#
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15 b b 2#
bc c b 3#
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5 4#

0
D1 18:00 D2 6:00 D2 18:00 D3 6:00 D3 18:00
Time

(c)
Figure 5. (a) The DIC of modes at different times. The different letters indicate a significant
Figure 5. (a) The DIC of modes at different times. The different letters indicate a significant difference
difference of DIC within mode at different detecting times. Means ± SE (n = 3). (b) The fluctuations
of
of DIC
pCO2within mode
in modes at different
at different detecting
times. times.letters
The different Means ± SE a(nsignificant
indicate = 3). (b) The fluctuations
difference of pCOof
2
pCO2 in
modes at different times. The different letters indicate a significant difference of pCO2 within mode
at different detecting times. Means ± SE (n = 3). (c) The variation of carbonate system in modes
at different times: (ci) HCO3 − ; (cii) CO3 2− ; (ciii) CO2 . The different letters indicate a significant
difference of HCO3 − , CO3 2− , and CO2 within modes at different detecting times. Means ± SE (n = 3).
21, 6, x FOR PEER REVIEW
Fishes 2021, 6, 53 11 of 16

1.8
a
1.6
a a
a
1.4 a a a
b
a b b
1.2 ab
b a c cd b bcbd bc
0#
POC (mg/L)

ab
1 b ac
1#
0.8 bcd d
2#
0.6
3#
0.4
4#
0.2
0
D1 18:00 D2 6:00 D2 18:00 D3 6:00 D3 18:00
Time

(a)
1200
a
1000 aa
ca c c c cd b bbb b bbb ad
c d b c
c
800 0#
DOC (μmol/L)

600 1#
2#
400
3#
200
4#
0
D1 18:00 D2 6:00 D2 18:00 D3 6:00 D3 18:00
Time

(b)
Figure
gure 6. (a) The POC6. (a)of
Themodes
POC of modes at different
at different times. The
times. Thedifferent lettersletters
different indicate indicate
a significantadifference of POC
significant within modes
difference of POC with
at different detecting times. Means ± SE (n = 3). (b) The DOC of modes at different times. The different letters indicate a
odes at different detecting times. Means ± SE (n = 3). (b) The DOC of modes at different times. The different lette
significant difference of DOC within modes at different detecting times. Means ± SE (n = 3).
dicate a significant difference of DOC within modes at different detecting times. Means ± SE (n = 3).

Table 5. Two-way repeated ANOVA results of POC and DOC in the seawater of modes at different sampling times.

Source Sum of Squares df Mean Squares F p

Mode 48,740.061 4 12,185.015 50.286 0.0
Fishes 2021, 6, 53 12 of 16

Table 6. The nutrient concentrations and structural changes in the seawater of modes at the beginning and at the end of
the experiment.

Mode Time SiO4 DIN PO4 Si:P N:P Si:N

All gourps Initial 14.88 ± 2.35 16.04 ± 1.32 1.57 ± 0.27 9.48 10.22 0.93
0# Final 14.32 ± 1.22 3.85 ± 0.87 0.48 ± 0.12 29.83 8.02 3.72
1# Final 6.56 ± 1.01 18.86 ± 2.21 1.32 ± 0.24 4.97 14.29 0.35
2# Final 14.77 ± 1.25 2.37 ± 1.06 0.30 ± 0.11 36.93 7.9 6.23
3# Final 8.21 ± 0.97 9.64 ± 1.13 0.49 ± 0.25 16.76 19.67 0.60
4# Final 8.94 ± 1.33 8.9 ± 1.21 0.34 ± 0.10 26.29 26.17 0.69

4. Discussion
In summer, when much of the kelp is harvested under high temperatures, the main
issue of shellfish culture becomes the impact of the substances released and excreted
during their growth process on the aquatic environment. In our study, the pH and DO were
significantly reduced (p < 0.05) by the end of the experiment in group 1, where shellfish
were cultured alone, and the DO concentration had dropped to below 5 mg/L. The increase
in DIN concentration also demonstrated how shellfish cultures can exacerbate the risk
of eutrophication. Zhang [20] has reported that shellfish were unable to form shells at
pH 7.3, at DO levels of 4–5 mg/L where the growth of fish was inhibited and some even
died, and at DO of 3–4 mg/L where the mass mortality of crustaceans can occur [21–23].
Therefore, if the shellfish culture scale is large, mismanagement may easily bring harm to
the environment in semienclosed areas or in areas with poor hydrodynamics. Therefore,
as during summer the common cocultivated algae, the species of kelp, cannot be grown
due to high water temperature, it is necessary to use macroalgal rotation strategies for
cocultures to avoid the negative effects of shellfish monocultures.
In the shellfish–algae IMTA areas, after the summer kelp is harvested, the kelp farming
area can be used to cultivate seaweed, which can improve environmental parameters and
fully absorb the nutrients in the water, thereby alleviating the environmental pressure
caused by the shellfish culture [24–28]. However, only the appropriate ratio of shellfish to
algae can achieve an environmentally friendly aquaculture system [7]. Generally, it seems
that the greater the proportion of algae, the greater the benefit to environmental indicators
(e.g., pH and DO) [2]. However, in low nutrient areas, or areas with poor water exchange,
cultivation with too large a proportion of algae may lead to the excessive consumption of
nutrients such as DIC, N, and P [29]. The same conclusion was reached in our study. At
the end of the experiment, the DIC of group 2 with only algae was significantly reduced
and the DIN was nearing the threshold of 2 µmol/L at which point N becomes a limiting
factor for phytoplankton growth [30,31]. In contrast, at the end of the experiment in the
shellfish–algae IMTA mode group, N:P:Si did not deviate from the Redfield ratio (N:P:Si =
16:1:16) [32] so that there was no potential limiting nutrient, thus demonstrating that the
shellfish–algae mode had little to no negative influence on the ambient nutrient structure.
Chl-a reflects the biomass of the phytoplankton, which is the main food source for
shellfish. The phytoplankton community faces downward controlling effects due to shell-
fish ingestion and upward controlling effects due to competition with macroalgae for
nutrients during growth [33,34]. In our study, when the ratio of shellfish to algae was 1:1,
the concentration of Chl-a at end of the experiment was significantly higher than that in
other experimental groups. This was because shellfish have phytoplankton size preferences
and actively select the size class on which they feed, and Japanese scallop mainly feeds on
nanophytoplankton (2–20 µm) [35,36]. This meant that the growth of microphytoplankton
(>20 µm) and picophytoplankton (0.45–2 µm) were not impeded in this shellfish–algae
system. In order to avoid eutrophication and food shortages for shellfish, the Chl-a con-
centration should be maintained at a stable level. Furthermore, it appeared that in the
actual culture process if the proportion of algae is too low the upward controlling effect of
Fishes 2021, 6, 53 13 of 16

macroalgae on nutrient competition was not strong, which could lead to overpopulating
by picophytoplankton and microphytoplankton.
Under high temperature conditions the respiration rate of scallops and CO2 production
increased but the IMTA mode was able to effectively absorb the nutrients released by the
shellfish and thereby prevent the accumulation of CO2 produced by respiration. In the
shellfish–algae IMTA mode, 70% of the CO2 released by shellfish can be absorbed by
algae [8], making the aquaculture system a net carbon sink [7]. Han [37] reported that
when the Chlamys farreri to seaweed ratio exceeded 1:0.31, the inorganic carbon system
in the water was improved. Likewise, this study found that the DIC, carbonate system
components, and the pCO2 were all significantly reduced in the shellfish–algae polyculture
group. We also found that the greater the proportion of algae, the more significant the
impact on the inorganic carbon cycle. It should be noted that cultivation with too large
a proportion of algae might lead to the excessive consumption of nutrients, and that the
pursuit of improving the inorganic carbon system of the water body needs to be tempered
by the awareness of other environmental parameters. Our study verified the importance
of the appropriate culture ratios in practical production. At the end of the experiment,
when the ratio of shellfish to algae was 1:1.6, the pCO2 and the inorganic carbon system
components were not significantly different from those in group 2 where the algae were
cultured alone. Therefore, it seems possible to assume that the most significant effect on
the inorganic carbon cycle was observed in summer when the ratio of Japanese scallop
to seaweed was 1:1.6 under experimental conditions. However, in nature there is water
exchange in areas occupied by aquaculture, and the real level of water quality parameters
may differ significantly from those obtained in a closed experimental system. The optimal
ratio of scallop to algae may also differ significantly from that which is experimentally
determined in a closed system. Further large-scale field studies are needed to assess the
optimal scallop and algae ratio.
Although shellfish–algae farming influences the inorganic carbon system in the water,
the “inorganic carbon pump” only enables the transport of CO2 from the atmospheric
carbon reservoir to the oceanic carbon reservoir. For the long-term storage of CO2 , the CO2
entering the ocean must be entrained by the “biological carbon pump” [38]. Therefore,
the cycling of organic carbon by the aquaculture process is important and should also
be considered. Although CO2 emissions from shellfish respiration do not affect the TA
(i.e., the ability to neutralize hydrogen ions) in seawater [20], calcification by shellfish
decreases the TA, which limits the ability of seawater to absorb CO2 and further affects the
carbonate chemistry equilibrium [39]. Therefore, if we only look at the direct evidence of
inorganic carbon system parameters in the water, shellfish farming should be a source of
carbon. However, it has been established that shellfish aquaculture can be a carbon sink,
not only via absorbing the DIC and synthesizing the shells, but also through its efficient
water filtration and biological activities, which can effectively filter out particulate carbon
and transport it to the sediment [6,8]. In our study, it was found that the POC and the
DOC had decreased significantly by the end of the experiment in group 1, which had only
shellfish cultures. Studies on the shellfish carbon balance have found that 30% of the carbon
used by shellfish during a growth cycle was retained by the shellfish itself, 30% sunk to the
bottom of the sea as waste, and 30–40% was released by the respiration and calcification
processes [40,41]. Therefore, the controversy around whether shellfish aquaculture is a
carbon source or sink should not only be based only on the inorganic carbon cycle but
should include more detailed studies that track inorganic and organic carbon as well as the
physiological processes of the shellfish themselves. Comprehensive studies such as this
one may provide insight into the controversial issue of carbon sequestration in shellfish.
Additionally, in the groups containing algae, the DOC content significantly increased. The
DOC makes up a portion of organic carbon that is effectively sequestered as it enters food
webs via the action of microfood loops or forms inert organic carbon and stays in the sea
for a long time [42,43]. According to Jiao [44], microbial carbon sequestration as a carbon
sink is often overlooked, and the core mechanism of microbial carbon sequestration is its
Fishes 2021, 6, 53 14 of 16

conversion of DOC into refractory DOC (RDOC) that is stored in seawater for extended
periods. Therefore, the organic carbon cycle also plays an important role as a carbon sink
in aquaculture systems. The shellfish–algae IMTA mode not only changes the inorganic
carbon system, but also effectively changes the organic carbon system, thus increasing the
function and role of aquaculture as a carbon sink.

5. Conclusions
In summer the respiratory efficiency of shellfish increases due to high temperatures,
and so large-scale shellfish cultures can easily harm environments in semienclosed areas
or in areas with poor hydrodynamics. Therefore, it is necessary to utilize macroalgae
rotation cultivation strategies in these areas in summer so to alleviate the effects of shellfish
monocultures. This study has shown that the summertime rotational aquaculture with
macroalgae benefited both the environment and the growth of shellfish. The shellfish–algae
IMTA mode not only mutually benefitted the shellfish and algae, but also positively affected
carbon sequestration. Moreover, the shellfish–algae IMTA mode not only altered the
inorganic carbon system, but also effectively changed the organic carbon system. However,
only the appropriate ratio of shellfish to algae can achieve an environmentally friendly
aquaculture system. Since this experiment was conducted in an enclosed environment, it is
necessary to further investigate and verify these results on a larger scale in the aquaculture
area in order to extend these findings to the production level. Such experiments will
provide supportive information for making governmental policies that guide the rotational
aquaculture of macroalgae in summer, as well as provide data that may help resolve the
controversy around aquaculture as a carbon sink.

Author Contributions: Conceptualization, Y.L. and J.Z.; methodology, Y.L.; experimental manipula-
tion, W.W.; sample analysis, W.W. and J.Y.; data curation, X.W.; writing—original draft preparation,
Y.L.; writing—review and editing, Y.L. and N.W. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by The National Key R&D Program of China: 2020YFA0607603,
2020YFA0607602. Joint Fund of National Natural Science Foundation of China: U1906216. Strategic
Priority Research Program of the Chinese Academy of Sciences: XDA23050402. Ministry of agricul-
ture national outstanding agricultural talents and innovative team “shallow aquaculture capacity
and healthy aquaculture”.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The datasets used during and/or analyzed during the current study
are available from the corresponding authors on reasonable request.
Acknowledgments: We would like to thank editors and anonymous reviewrers substantially im-
proved earlier versions of this paper, and Shandong Rongcheng Xunshan Group for providing
experimental shellfish and algae.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Zhang, Y. Comparison of Culture Effect, Discharge of Nitrogen and Phosphorus and Environmental Influence for Three Kinds of
Cages. Master’s Thesis, Huazhong Agriculture University, Wuhan, China, 2012. (In Chinese with English abstract).
2. Fang, J.; Fang, J.; Chen, Q.; Mao, Y.; Jiang, Z.; Du, M.; Gao, Y.; Lin, F. Assessing the effects of oyster/kelp weight ratio on water
column properties: An experimental IMTA study at Sanggou Bay, China. J. Oceanol. Limnol. 2019, 38, 3–4. [CrossRef]
3. Reid, G.K.; Lefebvre, S.; Filgueira, R.; Robinson, S.M.C.; Broch, O.J.; Dumas, A.; Chopin, T.B.R. Performance measures and models
for open-water integrated multi-trophic aqua-culture. Rev. Aquac. 2018, 12, 47–75. [CrossRef]
4. Troell, M.; Halling, C.; Neori, A.; Chopin, T.; Buschmann, A.; Kautsky, N.; Yarish, C. Integrated mariculture: Asking the right
questions. Aquaculture 2003, 226, 69–90. [CrossRef]
5. Tang, Q.S.; Fang, J.G.; Zhang, J.H.; Jiang, Z.J.; Liu, H.M. Impacts of multiple stressors on coastal ocean ecosystems and integrated
multi-trophic aquaculture. Prog. Fish. Sci. 2013, 34, 1–11, (In Chinese with English abstract).
Fishes 2021, 6, 53 15 of 16

6. Tang, Q.; Zhang, J.; Fang, J. Shellfish and seaweed mariculture increase atmospheric CO2 absorption by coastal ecosystems. Mar.
Ecol. Prog. Ser. 2011, 424, 97–104. [CrossRef]
7. Jiang, W.; Fang, J. Effects of mussel-kelp ratios in integrated mariculture on the carbon dioxide system in Sanggou Bay. J. Sea Res.
2021, 167, 101983. [CrossRef]
8. Jiang, Z.; Wang, G.; Fang, J.; Mao, Y. Growth and food sources of Pacific oyster Crassostrea gigas integrated culture with Sea bass
Lateolabrax japonicus in Ailian Bay, China. Aquac. Int. 2012, 21, 45–52. [CrossRef]
9. Chopin, T.; Robinson, S.; Troell, M.; Neori, A.; Buschmann, A.; Fang, J. Multitrophic Integration for Sustainable Marine
Aquaculture. Encycl. Ecol. 2008, 2463–2475. [CrossRef]
10. Ning, Z.; Liu, S.; Zhang, G.; Ning, X.; Li, R.; Jiang, Z.; Fang, J.; Zhang, J. Impacts of an integrated multi-trophic aquaculture
system on benthic nutrient fluxes: A case study in Sanggou Bay, China. Aquac. Environ. Interact. 2016, 8, 221–232. [CrossRef]
11. Chauvaud, L.; Thompson, J.K.; Cloern, J.; Thouzeau, G. Clams as CO2 generators: The Potamocorbula amurensis example in San
Francisco Bay. Limnol. Oceanogr. 2003, 48, 2086–2092. [CrossRef]
12. Suikkanen, S.; Silvia, P.; Engström-Öst, J.; Lehtiniemi, M.; Lehtinen, S.; Brutemark, A. Climate Change and Eutrophication
Induced Shifts in Northern Summer Plankton Communities. PLoS ONE 2013, 8, e66475. [CrossRef]
13. Joseph, S.S.; Winston, R.J.; Tirpak, R.A.; Wituszynski, D.M.; Boening, K.M.; Martin, J.F. The seasonality of nutrients and sediment
in residential stormwater runoff: Impli-cations for nutrient-sensitive waters. J. Environ. Manag. 2020, 276, 111248.
14. Su, J.; Cai, W.-J.; Brodeur, J.; Chen, B.; Hussain, N.; Yao, Y.; Ni, C.; Testa, J.M.; Li, M.; Xie, X.; et al. Chesapeake Bay acidification
buffered by spatially decoupled carbonate mineral cycling. Nat. Geosci. 2020, 13, 441–447. [CrossRef]
15. Nikinmaa, M. Climate change and ocean acidification—Interactions with aquatic toxicology. Aquat. Toxicol. 2013, 126, 365–372.
[CrossRef] [PubMed]
16. Kanerva, M.; Kiljunen, M.; Torniainen, J.; Nikinmaa, M.; Dutz, J.; Vuori, K.A. Environmentally driven changes in Baltic salmon
oxidative status during marine migration. Sci. Total Environ. 2020, 742, 140259. [CrossRef] [PubMed]
17. Zhou, Y.; Yang, H.; Hu, H.; Liu, Y.; Mao, Y.; Zhou, H.; Xu, X.; Zhang, F. Bioremediation potential of the macroalga Gracilaria
lemaneiformis (Rhodophyta) integrated into fed fish culture in coastal waters of north China. Aquaculture 2006, 252, 264–276.
[CrossRef]
18. Fei, X.G.; Lu, S.; Bao, Y.; Wilkes, R.; Yarish, C. Seaweed cultivation in China. World Aquac. 1998, 29, 22–24.
19. Fei, X.G.; Bao, Y.; Lu, S. Seaweed cultivation—Traditional way and its reformation. Aquac. Oceanol. Limnol. Sin. 1999, 17, 193–199.
20. Zhang, M.; Fang, J.; Zhang, J.; Bin, L.; Ren, S.; Mao, Y.; Gao, Y. Effect of Marine Acidification on Calcification and Respiration of
Chlamys farreri. J. Shellfish Res. 2011, 30, 267–271.
21. Sugden, A.M. Threats of coastal hypoxia. Science 2017, 356, 38. [CrossRef] [PubMed]
22. Levin, L.; Breitburg, D.L. Linking coasts and seas to address ocean deoxygenation. Nat. Clim. Chang. 2015, 5, 401–403. [CrossRef]
23. Vaquer-Sunyer, R.; Duarte, C.M. Thresholds of hypoxia for marine biodiversity. Proc. Natl. Acad. Sci. USA 2008, 105, 15452–15457.
[CrossRef]
24. Fang, J.H.; Zhang, J.H.; Wu, W.G.; Mao, Y.Z.; Gao, Y.P.; Jiang, Z.J.; Fang, J.G. Carbon and nitrogen budget and environmental
optimization in an integrated cage culture model of Japanese flounder with Perinereis aibuhitensis. J. Fish. Sci. 2014, 21, 390–397,
(In Chinese with English abstract).
25. Mao, Y.; Yang, H.; Zhou, Y.; Ye, N.; Fang, J. Potential of the seaweed Gracilaria lemaneiformis for integrated multi-trophic
aquaculture with scallop Chlamys farreri in North China. J. Appl. Phycol. 2009, 21, 649–656. [CrossRef]
26. Phillips, J.; Hurd, C. Nitrogen ecophysiology of intertidal seaweeds from New Zealand: N uptake, storage and utilisation in
relation to shore position and season. Mar. Ecol. Prog. Ser. 2003, 264, 31–48. [CrossRef]
27. Pedersen, A.; Kraemer, G.; Yarish, C. The effects of temperature and nutrient concentrations on nitrate and phosphate uptake in
different species of Porphyra from Long Island Sound (USA). J. Exp. Mar. Biol. Ecol. 2004, 312, 235–252. [CrossRef]
28. Lartigue, J.; Sherman, T.D. Response of Enteromorpha sp. (Chlorophyceae) to a nitrate pulse: Nitrate uptake, inorganic nitrogen
storage and nitrate reductase activity. Mar. Ecol. Prog. Ser. 2005, 292, 147–157. [CrossRef]
29. Zou, D.H.; Xia, J.R.; Yang, Y.F. Photosynthetic use of exogenous inorganic carbon in the agarophyte Gracilaria lemaneiformis
(Rhodophyta). Aquaculture 2004, 237, 421–431. [CrossRef]
30. Fisher, T.; Peele, E.; Ammerman, J.; Harding, L. Nutrient limitation of phytoplankton in Chesapeake Bay. Mar. Ecol. Prog. Ser.
1992, 82, 51–63. [CrossRef]
31. Justić, D.; Rabalais, N.N.; Turner, R.E.; Dortch, Q. Changes in nutrient structure of river-dominated coastal waters: Stoichiometric
nutrient balance and its consequences. Estuar. Coast. Shelf Sci. 1995, 40, 339–356. [CrossRef]
32. Redfield, A.C.; Ketchum, B.H.; Richard, F.A. The influence of organism on the composition of seawater. Sea 1963, 2, 26–77.
33. Prins, T.C.; Escaravage, V.; Smaal, A.C.; Peeters, J.C.H. Nutrient cycling and phytoplankton dynamics in relation to mussel
grazing in a mesocosm experiment. Ophelia 1995, 41, 289–315. [CrossRef]
34. Souchu, P.; Vaquer, A.; Collos, Y.; Landrein, S.; Deslous-Paoli, J.; Bibent, B. Influence of shellfish farming activities on the
biogeochemical composition of the water column in Thau lagoon. Mar. Ecol. Prog. Ser. 2001, 218, 141–152. [CrossRef]
35. Dupuy, C.; Vaquer, A.; Lam-Höai, T.; Rougier, C.; Mazouni, N.; Lautier, J.; Collos, Y.; Le Gall, S. Feeding rate of the oyster
Crassostrea gigas in a natural planktonic community of the Mediterranean Thau Lagoon. Mar. Ecol. Prog. Ser. 2000, 205, 171–184.
[CrossRef]
Fishes 2021, 6, 53 16 of 16

36. Lu, J.C.; Huang, L.F.; Xiao, T.; Jiang, Z.; Zhang, W. The effects of Zhikong scallop (Chlamys farreri) on the microbial food web in a
phospho-rus-deficient mariculture system in Sanggou Bay, China. Aquaculture 2015, 448, 341–349. [CrossRef]
37. Han, T.; Jiang, Z.; Fang, J.; Zhang, J.; Mao, Y.; Zou, J.; Huang, Y.; Wang, D. Carbon dioxide fixation by the seaweed Gracilaria
lemaneiformis in integrated multi-trophic aquaculture with the scallop Chlamys farreri in Sanggou Bay, China. Aquac. Int. 2013,
21, 1035–1043. [CrossRef]
38. Schlitzer, R. Export and sequestration of particulate organic carbon in the north pacific from inverse modeling. In Proceedings of
the Workshop of the JGOFS North Pacific Process Study Synthesis Group, Sapporo, Japan, 2 October 2002.
39. Han, T.; Shi, R.; Qi, Z.; Huang, H.; Wu, F.; Gong, X. Biogenic acidification of Portuguese oyster Magallana angulata mariculture
can be mediated through introducing brown seaweed Sargassum hemiphyllum. Aquaculture 2020, 520, 734972. [CrossRef]
40. Ren, L.H.; Zhang, J.H.; Fang, J.G.; Tang, Q.H.; Liu, Y.; Du, M.R. The diurnal rhythm of respiration, excretion and calcification in
oyster Crassostre gigas. Prog. Fish. Sci. 2013, 1, 75–81, (In Chinese with English abstract).
41. Zhang, J.H.; Fang, J.G.; Tang, Q.S.; Ren, L.H. Carbon sequestration rate of the scallop Chlamys farreri cultivated in different areas
of Sanggou Bay. Prog. Fish. Sci. 2013, 34, 12–16, (In Chinese with English abstract).
42. Zhang, Y.; Zhang, J.; Liang, Y.; Li, H.; Li, G.; Chen, X.; Zhao, P.; Jiang, Z.; Zou, D.; Liu, X.; et al. Carbon sequestration processes
and mechanisms in coastal mariculture environments in China. Sci. China Earth Sci. 2017, 60, 2097–2107. [CrossRef]
43. Li, H.; Zhang, Y.; Liang, Y.; Chen, J.; Zhu, Y.; Zhao, Y.; Jiao, N. Impacts of maricultural activities on characteristics of dissolved
organic carbon and nutrients in a typical raft-culture area of the Yellow Sea, North China. Mar. Pollut. Bull. 2018, 137, 456–464.
[CrossRef] [PubMed]
44. Jiao, N.; Cai, R.; Zheng, Q.; Tang, K.; Liu, J.; Jiao, F.; Wallace, D.; Chen, F.; Li, C.; Amann, R.; et al. Unveiling the enigma of
refractory carbon in the ocean. Natl. Sci. Rev. 2018, 5, 459–463. [CrossRef]