appliedsciences
ArticleDistribution of Harmful Algal Growth-Limiting
Bacteria on Artificially Introduced Ulva and Natural
Macroalgal Beds
Nobuharu Inaba 1, * , Isamu Kodama 2 , Satoshi Nagai 3 , Tomotaka Shiraishi 4 ,Kohei Matsuno 2,5 , Atsushi Yamaguchi 2,5 and Ichiro Imai 2
1
2

3
4
5

*

Civil Engineering Research Institute for Cold Region, Public Works Research Institute, Sapporo City,
Hokkaido 062-8602, Japan
Faculty/Graduate School of Fisheries Sciences, Hokkaido University, Hakodate City, Hokkaido 041-8611,
Japan; k-damakan@fish.hokudai.ac.jp (I.K.); k.matsuno@fish.hokudai.ac.jp (K.M.);a-yama@fish.hokudai.ac.jp (A.Y.); imai1ro@fish.hokudai.ac.jp (I.I.)
National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency,
Yokohama City, Kanagawa 236-8648, Japan; snagai@affrc.go.jp
Agriculture, Forestry and Fisheries Department, Wakayama Prefecture, Wakayama City,
Wakayama 640-8585, Japan; shiraishi_t0002@pref.wakayama.lg.jp
Arctic Research Center, Hokkaido University, Sapporo City, Hokkaido 001-0021, Japan
Correspondence: inaba-n@ceri.go.jp; Tel.: +81-11-841-1695


Received: 8 July 2020; Accepted: 12 August 2020; Published: 14 August 2020

Abstract: The intensity and frequency of harmful algal blooms (HABs) have increased, posing a threat
to human seafood resources due to massive kills of cultured fish and toxin contamination of bivalves.
In recent years, bacteria that inhibit the growth of HAB species were found to be densely populatedon the biofilms of some macroalgal species, indicating the possible biological control of HABs by
the artificial introduction of macroalgal beds. In this study, an artificially created Ulva pertusa bed
using mobile floating cages and a natural macroalgal bed were studied to elucidate the distributionof algal growth-limiting bacteria (GLB). The density of GLB affecting fish-killing raphidophyte
Chattonella antiqua, and two harmful dinoflagellates, were detected between 106 and 107 CFU g−1
wet weight on the biofilm of artificially introduced U. pertusa and 10 to 102 CFU mL−1 from adjacentseawater; however, GLB found from natural macroalgal species targeted all tested HAB species (fivespecies), ranging between 105 and 106 CFU g−1 wet weight in density. These findings provide new
ecological insights of GLB at macroalgal beds, and concurrently demonstrate the possible biologicalcontrol of HABs by artificially introduced Ulva beds.
Keywords: harmful algal bloom; mitigation; macroalgal bed; growth-limiting bacteria

1. Introduction
Harmful algal blooms (HABs) are naturally-occurring phenomena in fresh and marine water
systems, often causing severe damage to the fishery industry worldwide, through massive kills ofcommercially cultured fishes and toxin-contamination of shellfish leading to closures of commercial
shellfish harvests, and, in some cases, consumption of contaminated shellfish results in human illness
and even death [1–3]. Lately, the acceleration of geographic expansion and intensity of HABs on a
global scale associated with climate change have been reported [4–7]. Hence, developments of effective
countermeasures to control the HAB events are even more urgent and crucial concerns. Mechanical,
chemical, genetic, biological, and environmental measures to prevent HAB have been proposed [4],and clay dispersion is the only practical measure confirmed to control HABs formed by Cochlodinium
Appl. Sci. 2020, 10, 5658; doi:10.3390/app10165658

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polykrikoides in Korean coastal waters [8]. However, the negative impacts of clay on non-target
organisms living in the seafloor and water column are also pointed out [9–12]. The control of HABs
directly intervenes with surroundings in dynamic ocean environments, demanding an establishmentof measures as environmentally friendly as possible. Among all proposed strategies to mitigate HABs,
biological controls using naturally living organisms may have the least impact on ecosystems in thepresent circumstances. Numbers of biological controls using microbes to bivalves have been proposed
and tested extensively for several decades [13–20]. In particular, bacteria that kill and/or inhibit thegrowth of HAB species often associated with bloom termination have received worldwide attention as
possible biological control of HABs [21–31].
Although the use of natural pathogenicity could be environmentally friendly and cost-effective tocontrol HABs, introducing potentially invasive bacteria may require a careful manner [32,33]. Recently,
a biofilm of seagrass and macroalgae were found to be densely populated by algal growth-limiting
bacteria (GLB), including both algicidal and growth-inhibiting bacteria [34–37], indicating theirfunction as an enormous source of GLB for the adjacent seawater [31,38]. The latest microcosm study
demonstrated that seawater in the Zostera marina bed successfully suppressed artificial Chattonella
blooms [31], indicating that protection and restoration of seagrass and macroalgal beds prevent theoutbreak of HABs through providing the specific microbial communities. However, these holistic
approaches may sometimes not be flexible in actual field applications in that they often require specifichabitat characteristics such as light, sediment or substrate types, currents, bioturbation, etc. [39,40].
In this study, Ulva pertusa beds using mobile floating cages were artificially created to study the
distribution of algal growth-limiting bacteria (GLB), seeking flexible and widely applicable technologies
to control HABs. A natural macroalgal bed was also studied for comparison.
2. Materials and Methods
2.1. Installation of Artificial Ulva Bed
Yellowish-green reproductively matured Ulva pertusa thalli were provided from Wakayama
prefectural fisheries experiment station, Wakayama Prefecture, Japan. After the thallus of U. pertusa
was gently wiped and dried in the shade for 1–2 h, it was placed into a water tank filled with
autoclaved-filtered seawater [41]. Pumice stones were carefully rinsed with autoclaved seawater andgently set to promote attachments of motile zoospore of U. pertusa [42]. Then, the pumice stones wereplaced into mobile floating cages and installed on June 20, 2017, in a nearshore area by an uprightseawall at Station 1 (Stn. 1), Kushimoto-cho, Wakayama Prefecture, Japan (Figure 1). The location of
installation was carefully selected after confirming the absence of any macroalgal species in the area.
However, this may not guarantee attachments of naturally-occurring macroalgal propagules to thetested pumice stone and cages after installation.
2.2. Sampling
Samplings were performed on 7 to 8 September 2017, at Stn. 1 (artificial Ulva bed) and Stn. 2(a natural macroalgal bed), Taiji-cho, Wakayama Prefecture, Japan (Figure 1). The weather was mostly
cloudy during the sampling on 7 September 2017, with surface seawater temperature and salinity of
27.9 ◦ C, 34.0 PSU at Stn. 1. On 8 September 2017, it was sunny with surface seawater temperatureand salinity of 29.2 ◦ C, 34.7 PSU at Stn. 2. The green alga, U. pertusa, successfully grown on the
pumice stone and floating cage at Stn. 1 (Figure 2) and four dominant macroalgae (one brown alga:
Sargassum dupulicatum, one red alga: Gelidium elegans, two green algae: Cladophora ohkuboana andU. pertusa) at Stn. 2 were sampled using sterile instruments in a sterile manner. Adjacent surface
seawater samples were also collected from both sites. Samplings were conducted at low tide, and all
the samples were collected in an autoclaved polycarbonate bottle (500 mL) filled with autoclaved,
filtered (0.7 µm GF/F) seawater. All the samples were immediately transported to the laboratory andprocessed on the same day.

autoclaved-filtered seawater [41]. Pumice stones were carefully rinsed with autoclaved seawater andgently set to promote attachments of motile zoospore of U. pertusa [42]. Then, the pumice stones wereplaced into mobile floating cages and installed on June 20, 2017, in a nearshore area by an uprightseawall at Station 1 (Stn. 1), Kushimoto-cho, Wakayama Prefecture, Japan (Figure 1). The location of
installation was carefully selected after confirming the absence of any macroalgal species in the area.
Appl. Sci. 2020, 10, 5658
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However, this may not guarantee attachments of naturally-occurring macroalgal propagules to thetested pumice stone and cages after installation.
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3 of 162.2. Sampling
Samplings were performed on 7 to 8 September 2017, at Stn. 1 (artificial Ulva bed) and Stn. 2 (anatural macroalgal bed), Taiji-cho, Wakayama Prefecture, Japan (Figure 1). The weather was mostly
cloudy during the sampling on 7 September 2017, with surface seawater temperature and salinity of
27.9 ℃, 34.0 PSU at Stn. 1. On 8 September 2017, it was sunny with surface seawater temperature andsalinity of 29.2 ℃, 34.7 PSU at Stn. 2. The green alga, U. pertusa, successfully grown on the pumicestone and floating cage at Stn. 1 (Figure 2) and four dominant macroalgae (one brown alga: Sargassum
dupulicatum, one red alga: Gelidium elegans, two green algae: Cladophora ohkuboana and U. pertusa) at
Stn. 2 were sampled using sterile instruments in a sterile manner. Adjacent surface seawater samples
were also collected from both sites. Samplings were conducted at low tide, and all the samples werecollected in an autoclaved polycarbonate bottle (500 mL) filled with autoclaved, filtered (0.7 µm GF/F)
Figure1.1.Location
Locationofofthe
theinstallation
installationofofartificial
artificialUlva
Ulvabedbed(Stn.
(Stn.1)1)andandthethenatural
naturalmacroalgal
macroalgalbed
bed(Stn.
(Stn.
Figureseawater. All the samples were immediately transported to the laboratory and processed on the same2)atatKushimoto-cho
Kushimoto-choandandTaiji-cho,Taiji-cho, Wakayama
Wakayama Prefecture, Japan.
2)
day.

FigureFigure 2. Pictures
Pictures ofof artificiallyartificially introduced
introduced (a)(a) Ulva
Ulva beds
beds using
using floating
floating cages
cages at Stn. 1;
1; (b)(b) aa landed
landedfloating cage U. pertusa is well grown; (c) U. pertusa grew on the pumice stone.
floating

2.3. Sample
Sample Processing
Processing andand Bacterial
Bacterial Culturing
Culturing
2.3.
Macroalgal samples
samples using
using bottles
bottles filled
filled with
with 200
200 mLmL autoclaved
autoclaved filteredfiltered seawater
seawater were
were shaken
shakenMacroalgal
600
times
by
hand
using
a
hand
tally
countertodetachabiofilm
formedonthesurfaceofthemacroalgae.
600 times by hand using a hand tally counter to detach a biofilm formed on the surface of themacroalgae. Biofilm-suspended samples were serially diluted 10-fold to 10−4 with autoclaved filteredseawater, and an aliquot (0.1 mL) of each dilution was spread onto a ST10 −1 marine agar plate(Trypticase peptone 0.5 g, Yeast extract 0.05 g and agar 15 g in 1 L seawater [43]). The adjacentseawater, also collected from both sites, were serially diluted 10-fold to 10−4, and an aliquot (1 mL)
was filtered through autoclaved 3 µm pore size Nucleopore Track-Etch membrane filters (Whatman,

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Biofilm-suspended samples were serially diluted 10-fold to 10−4 with autoclaved filtered seawater,
and an aliquot (0.1 mL) of each dilution was spread onto a ST10−1 marine agar plate (Trypticase
peptone 0.5 g, Yeast extract 0.05 g and agar 15 g in 1 L seawater [43]). The adjacent seawater, alsocollected from both sites, were serially diluted 10-fold to 10−4 , and an aliquot (1 mL) was filteredthrough autoclaved 3 µm pore size Nucleopore Track-Etch membrane filters (Whatman, GE Healthcare,
Parramatta, Australia). The filter was gently placed on a ST10−1 marine agar plate to culture theparticle-associated bacteria (PAB). An aliquot of 0.1 mL from the filtrate was also used to culture thefree-living bacteria (FLB) on the same medium above [44]. All the plates were kept for two weeks inthe dark at 20 ◦ C and used for the culturable bacterial enumeration and isolation. For the isolation,colonies were randomly picked by sterilized toothpicks from the agar plate, inoculated onto a 48-wellfilled with the same bacterial medium and kept at 20 ◦ C in the dark until the following co-culture
experiment. Aliquots (10 mL) from all samples were fixed with glutaraldehyde (1% final concentration)and stored at 4 ◦ C for later determination of total bacterial abundance by DAPI staining [45].
2.4. Targeted Harmful Algal Bloom (HAB) Species
Two raphidophycean flagellates, Chattonella antiqua NIES-1 and Heterosigma akashiwo 893, twoarmored dinoflagellates, Alexandrium catenella, and Heterocapsa circularisquama and one unarmoreddinoflagellate, Karenia mikimotoi, were axenically grown and kept in modified SWM-3 medium [46,47].
The cultured temperature was 15 ◦ C for A. catenella, 20 ◦ C for H. akashiwo and 25 ◦ C for C. antiqua,
H. circularisquama and K. mikimotoi under a light intensity of 50–100 µmol photons m−2 s−1 using a
14:10 h light–dark photocycle in algal growth chambers. Early stationary growth phase cultures wereused for following co-culture experiments to test bacterial growth inhibitory activities.
2.5. Co-Culture Experiment Using Bacterial Isolates
Cultured bacteria isolated from the biofilm of U. pertusa grown on the pumice stone and floating
cage (Stn. 1) and four macroalgal species (Stn. 2), as well as PAB and FLB from the surrounding seawaterat both sites, had their growth inhibitory properties determined through co-culturing experiments(a single axenic HAB species cultured in the algal medium with a single bacterium) toward fivetargeted HAB species [37]. Cell densities were diluted with the same algal medium to approximately
103 cells mL−1 for C. antiqua, A. catenella, and K. mikimotoi and 104 cells mL−1 for H. akashiwo andH. circularisquama, then 0.8 mL aliquots were incubated for two days in sterilized disposable 48-well
microplates to check for any abnormal appearance of cells. Individual bacterial isolates were inoculated
into the algal cultures with an approximate density of 105 cells mL−1 , and the plates were incubatedfor another two weeks with the conditions described above for the algal cultures. Bacteria-free
quadruplicate wells (Controls) were also prepared in each microplate. A Nikon ECLIPSE TE200
inverted microscope was used for daily checks of algal cell mortality and/or any abnormalities of algal
cells. The wells with 90% or more of algal cells killed were labeled as algicidal bacteria (AB), and thewells with 90% or more of motility reduced algal cells simultaneously were labeled as growth-inhibiting
bacteria (GIB). In this study, the term algal growth-limiting bacteria (GLB) was mainly applied toexpress a set of bacteria limiting algal growth, including both AB and GIB, to eliminate repeatedusage of those terms. The density of AB and GIB was estimated using the following formula: NAG =NV × SAG/NT (NAG is the number of AB or GIB (CFU mL−1 or CFU g−1 wet weight of macroalgae),
NV is the number of viable bacteria (CFU mL−1 or CFU g−1 wet weight of macroalgae), SAG is thenumber of bacterial strains that expressed growth inhibitory properties, NT is the number of bacterial
strains examined). All of the GLB were isolated aseptically into a 48-well plate after confirming a pureculture by the streak plate method [48].
2.6. 16S rRNA Gene Sequencing for Identification
A total of 47 bacterial strains (12 strains from Stn. 1 and 35 strains from Stn. 2) were identified bypartial 16S rRNA gene sequencing (500 bp). All strains were cultured in ST10−1 liquid medium for two

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weeks then centrifuged (8000 rpm) for 5 min to obtain bacterial pellets. Pellets were suspended inphosphate-buffered saline and washed three times to remove the medium. Bacterial DNA was extractedusing NucleoSpin Tissue XS (TaKaRa BIO Inc., Japan) and stored at −20 ◦ C. The universal primers 27F(50 -AGAGTTTGATCMTGGCTCAG-30 [49]) and 519R (50 -GWATTACCGCGGCKGCTG-30 [50]) wereapplied to amplify the 16S rRNA gene fragments using Blend Taq® -Plus- (TOYOBO, Japan). The PCRtemperature cycling conditions were: initial denaturation at 94 ◦ C for 1 min, followed by 30 cycles
of denaturation at 94 ◦ C for 30 s, annealing at 52 ◦ C for 30 s, and extension at 72 ◦ C for 1 min; final
elongation was for 7 min at 72 ◦ C. PCR products were purified using ISOSPIN PCR Product (NIPPONGENE CO., LTD., Japan) before sequencing. Both strands of the PCR products were sequenced using
the same primers in a cycle sequencing reaction using a sequencing kit (Big Dye Terminator Cycle
version 3.1, Applied Biosystems, USA) using a DNA sequencer (ABI 3130, Applied Biosystems, USA).
Nucleotide sequences obtained were aligned using Chromas Pro (ver. 1.7.1). Approximately 500 bp ofeach consensus sequence were analyzed by A BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) sequencesimilarity search to identify similarity with previously reported bacteria.
3. Results
3.1. Culturable and Total Bacterial Enumeration
The densities of culturable and total bacteria from seawater and macroalgal biofilms are shown in
Table 1 and Figure 3. The densities of culturable free-living bacteria (FLB) at both sites were higher
than particle-associated bacteria (PAB) with values of 1.7 × 103 CFU mL−1 at Stn. 1 and 2.3 × 103 CFU
mL−1 at Stn. 2, respectively (Figure 3a). The density of culturable Ulva pertusa biofilm-associatedbacteria on the pumice stone (2.4 × 108 CFU g−1 wet weight) was about ten times higher than U. pertusa
collected from the floating cage (1.9 × 107 CFU g−1 wet weight) (Figure 3b). Sargassum dupulicatum
associated culturable bacteria showed the lowest density with 7.9 × 106 CFU g−1 wet weight, and thedensity of U. pertusa-associated culturable bacteria was the highest with 4.0 × 107 CFU g−1 wet weightat natural macroalgal bed (Stn. 2, Figure 3b). The density of total bacteria in seawater at Stn. 1 was
2.9 × 106 cells mL−1 (PAB: 2.6 × 106 cells mL−1 , FLB: 3.1 × 105 cells mL−1 ) higher than the density of
6.7 × 105 cells mL−1 (PAB: 3.9 × 105 cells mL−1 , FLB: 2.8 × 105 cells mL−1 ) at Stn.2 (Figure 3c). In contrast
to the culturable bacterial densities, the total PAB densities were higher than those of FLB at both sites(Figure 3a,c). The total bacterial density of U. pertusa on the pumice stone showed the maximum valueof 2.7 × 109 cells g−1 wet weight at Stn. 1 (Figure 3d) as well as culturable bacteria. Total bacterial
density of U. pertusa was the highest with the density of 1.2 × 109 cells g−1 wet weight at Stn.2 as well,followed by Cladophora ohkuboana (9.9 × 108 cells g−1 wet weight), Gelidium elegans (6.0 × 108 cells g−1
wet weight) and S. dupulicatum (3.9 × 108 cells g−1 wet weight) (Figure 3d). U. pertusa grown on the
pumice stone appeared to be unhealthy, turning partly black in decay, and resulted in the highest
values of both the densities of culturable and total bacteria at Stn. 1.
Table 1. Summary of culturable and total bacterial densities enumerated from seawater and macroalgal
biofilms at Stns. 1 and 2. (FLB: free-living bacteria, PAB: particle-associated bacteria)
Date

StationSample Type

Culturable Bacterial
Density (CFU mL−1 or
g−1 Wet Weight)

Total Bacterial Density(Cells mL−1 or g−1 Wet
Weight)FLB

1.7 × 103

3.1 × 105

PAB

1.0 × 103

2.6 × 106

Biofilm

2.4 × 108

2.7 × 109

Biofilm

1.9 × 107

1.7 × 109

FLB

2.3 × 103

2.8 × 105

PAB

1.8 × 103

3.9 × 105Sargassum dupulicatum

Biofilm

7.9 × 106

3.9 × 108

Gelidium elegans

Biofilm

1.5 × 107

6.0 × 108

U. pertusa

Biofilm

4.0 × 107

1.2 × 109

Biofilm

2.0 × 107

9.9 × 108

Sample Name

Seawater
2017/9/7

Stn. 1
Ulva pertusa on pumice stone
U. pertusa on floating cage
Seawater

2017/9/8

Stn. 2

Cladophora ohkuboana

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Figure 3. The densities of culturable bacteria (a): seawater, (b): macroalgal biofilms and total bacteria
Figure 3. The densities of culturable bacteria (a): seawater, (b): macroalgal biofilms and total bacteria
directly counted with DAPI staining (c): seawater, (d): macroalgal biofilms and at Stns. 1 and 2. Total
directly counted with DAPI staining (c): seawater, (d): macroalgal biofilms and at Stns. 1 and 2. Total(#) shown on (a) and (c) are expressed as the sum of particle-associated bacteria (PAB) and free-living(○) shown on (a) and (c) are expressed as the sum of particle-associated bacteria (PAB) and free-living
bacteria (FLB). A to F are macroalgal samples used to enumerate the densities.
bacteria (FLB). A to F are macroalgal samples used to enumerate the densities.

3.2. Density of Growth-Limiting Bacteria
3.2. Density of Growth-Limiting Bacteria
The densities of growth-limiting bacteria (GLB: the sum of algicidal bacteria (AB) and
The densities ofgrowth-limiting
bacteria(GLB:
the sumandof algicidal
bacteria(AB)atandgrowthgrowth-inhibiting
bacteria(GIB)) detectedfromseawater,macroalgal
samplesStns.1andinhibiting
bacteria(GIB))
detectedfromseawater,andmacroalgal
samplesatStns.1and2,
were
2, were shown in Table 2 and Figures 4 and 5. GLB targetting at least one HAB species were detected
shown
Table 2 andand Figures
4 and
5. GLBtargetting
at leastone HAB
species
were detectedboth inboth ininseawaterU. pertusa
samplescollected
at Stn.
1 (Figure4). Thedensities
of GLB showed
2 CFU mL
seawaterand against
U. pertusa
samplesantiquacollected
at3.5
Stn.
1 (Figure4).−1Thedensities
of×GLB
showedthatthat activities
Chattonella
were
× 10
in FLBand 1.9
102 CFU
mL−1
in
2 CFU mL−1 in FLB and 1.9 × 102 CFU mL−1 in PAB
activities
againstChattonella
antiqua
were
3.5
×
10
PAB (Figure 4a,b). GLB against Heterocapsa circularisquama and Karenia mikimotoi were detected only in−1 and 93 CFUand(Figure4a,b).
GLB against
Karenia
mikimotoi
were 4b).
detected
only in
PAB withdensities
of 1.9 × Heterocapsa
102 CFU mLcircularisquamamL−1, respectively(FigureAB targeted
2−1−1, respectively (Figure 4b). AB targeted C.
PAB
withdensities
of
1.9
×
10
CFU
mL
and
93
CFU
mL
C. antiqua, and H. circularisquama were detected from U. pertusa on the pumice stone with the same
antiqua,
andweredetectedfrom
pertusaon thestone withsame
density of
5.9H.× circularisquama
106 CFU g−1 wet
weight(Figure4c).U.GIB
targeted
H. pumicecircularisquama
wasthedetected
6−1
6−1
density
5.9 × 10g wet weight4c). GIB targeted
H. circularisquama
was detectedfrom U.of
pertusaon CFUthe floating
cage at (Figure
a concentrationof 1.5 × 10
CFU g wet weight(Figurefrom
4d).
6 CFU g−1 wet weight (Figure 4d). ThereU.
pertusaonthefloating
cage
ata
concentration
of
1.5
×
10
There were no GLB negatively affecting the growth of Alexandrium catenella and Heterosigma akashiwo
werenegatively
the growthof Alexandrium
andHeterosigma
akashiwo
at Stn.
at Stn.no1.GLB
GLB
targetingaffecting
at least one
HAB species
were alsocatenella
detectedfromall samples
collected
at1.
GLB
targeting
at
least
one
HAB
specieswere
also
detectedfromallsamplescollected
at
Stn.
2
Stn. 2 (Figure 5). The density of GLB showed that the activity against H. circularisquama was detected−1(Figure5).
Thedensityof
GLB
showedthattheactivity
againstH.
circularisquama
wasdetectedinFLB
in FLB with a density of 69 CFU mL (Figure 5a). GLB isolated from PAB at Stn. 2 had a wide−1
withdensityof 69
CFU mL5a). GLB
isolated
PABwith
at Stn.
2 had ranging
a wide range
ofrangeaof
activities
affectingfour (Figure
HAB species
exceptfor A.from
catenelladensities
between22−1
2−1
activities
affectingfour
HABspecies
exceptforA.
catenellawithdensities
ranging
between
1.1×
10
1.1 × 10 CFU mL and 7.9 × 10 CFU mL (Figure 5b). GLB affecting the growth of A. catenella,
2 CFU mL−1 (Figure 5b). GLB affecting the growth of A. catenella, H.
CFU
mL−1 and 7.9and× 10
H. circularisquama,
K. mikimotoi
were found from the S. dupulicatum biofilm with densities of
5
−1
6 CFU
circularisquama,
K. mikimotoi
were
found
from
dupulicatum
biofilm
4.0 ×
4.0 × 10 CFU gandwet
weight, 2.4
× 10
g−1thewetS.weightand 4.0
× 105with
CFUdensities
g−1 wet ofweight,5 CFU g−1 wet weight, 2.4 × 10 6 CFU g−1 wet weight and 4.0 × 10 5 CFU g−1 wet weight, respectively
10respectively (Figure 5c). GLB affecting C. antiqua, and H. circularisquama were detected from G. elegans
5 CFU and(Figure5c). GLB
affectingantiqua,
H. circularisquama
fromweight,
G. elegans
biofilm
biofilm withdensitiesof 7.6C.
× 10
g−1 wetweight and 1.5 were× 106 detected
CFU g−1 wet
respectively
5
−1
6
−1
with
densitiesof 7.6× 10 CFU
weight targetted
and 1.5 × four
10 CFUg species,
wet weight,
respectively(Figure(Figure5d). GLB
isolated
fromg C.wet
ohkuboana
HAB
exceptK. mikimotoi,
with
6
−1
5
−1
5d).
GLB
isolatedfromC.
ohkuboanatargetted
four
HAB
species,
exceptK.
mikimotoi,
withdensities
densities of 1.0 × 10 CFU g wet weight for A. catenella, 5.0 × 10 CFU g wet weight for C. antiqua,
6 CFU−1g−1 wet weight for A. catenella, 5.0 × 105 CFUof× 510CFU
g−1g−1wet
weightforforC.H.
antiqua,
5.0 × 105
5.01.0× 10
g wet weight for H. akashiwo, and 4.0 × 106 CFU
wetweightcircularisquama,−1
6−1
CFU g wet weight for H. akashiwo, and 4.0 × 10 CFU g wet weight for H. circularisquama,

Appl. Sci. 2020, 10, x FOR PEER REVIEW

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respectively (Figure 5e). GLB were found in U. pertusa with densities of 3.0 × 106 CFU g−1 wet weight
for A. catenella, 6.0 × 106 CFU g−1 wet weight for H. circularisquama and 2.0 × 106 CFU g−1 wet weightfor K. mikimotoi, respectively (Figure 5f). GLB negatively affecting the growth of all five HAB species
Appl. Sci. 2020, 10, 5658
7 of 16
were detected at the natural macroalgal bed (Stn. 2).
Table 2.(Figure
Summary
growth-limiting(GLB) withdensities
againstdifferent
harmfulalgal
respectively5e).ofGLB
were foundbacteriain U. pertusa
densities
of five
3.0 ×
106 CFU
g−1 wetweight
6
−1
6
−1
(HAB)
speciesestimatedthroughexperiments andfrom2.0
seawaterand gmacroalgal
for A.bloom
catenella,
6.0 ×
10 CFUg wetweightco-culturefor H. circularisquama× 10 CFU
wet weightat Stns.
1 and 2. (Figure 5f). GLB negatively affecting the growth of all five HAB species
for K.biofilms
mikimotoi,
respectively
were detected at the natural macroalgal bed (Stn.
2).density of GLB against five different HAB species
The
Date

StationSample(CFU mL−1 or g−1 wet weight)

Samplename
type
Alexandrium
Chattonella
Heterosigma
Heterocapsa
Karenia
Table 2. Summaryof growth-limiting
bacteria (GLB)
densities against
five different
harmful algal
catenella experiments
antiqua fromakashiwo
mikimotoi
bloom (HAB) species estimated through co-culture
seawater andcircularisquama
macroalgal biofilms
FLB
―
―
―
―
3.5 × 102
at Stns. 1 and 2.
SeawaterDate

2017/9/7

2017/9/7

PAB―― Five Different 1.9
93The
HAB×Species
1.9Density
× 102of GLB against
102
Appl. Sci. 2020, 10, x FOR PEER REVIEW
8 of 16(CFU mL−1 or g−1 Wet Weight)
SampleName
SampleType
Ulva pertusa
Stn. 1
Alexandrium
Chattonella
Heterosigma
Heterocapsa
Karenia
on pumice
Biofilm―catenella 5.9 ×―
antiquaakashiwo
circularisquama
mikimotoi―
106 of algicidal
5.9 × 106) andlimiting bacteria (●) are expressedas the sum
bacteria(AB:
growth-inhibiting
2
stone
FLB
—
—
—
—
3.5 × 10
Seawaterbacteria
(GIB:
).
The
testedHAB
speciesandtheirabbreviationsaredisplayedatthe bottom.
U. pertusa on
PAB
93
1.9 × 102
1.9 × 102
Biofilm― —――—―1.5 × 106
floating
cageonUlva pertusa
Station

Stn. 1

pumice stone
U. pertusa on
Seawaterfloating cage

Biofilm

FLB

Biofilm

PAB

―
―

FLB

2017/9/8
2017/9/8

Stn. 2
Stn. 2

SeawaterSargassum
PAB
Biofilm
dupulicatum
Sargassum
Biofilm
Gelidium
dupulicatum
Biofilm
elegans
Gelidium elegans
Biofilm
Cladophora
Cladophora
Biofilm
Biofilmohkuboana
ohkuboana

U. pertusa
U.REVIEW
pertusa
Appl. Sci. 2020, 10, x FOR PEER

Biofilm
Biofilm

5.9 × 106

—

―

—

—

3.4 × 102——5
4.0 × 10—

3.4 × 102―

4.0 × 105―—

5.9 × 106——

1.5 × 106

—

—

69

—

1.1 × 102
1.1
―× 102—

7.6 × 105 5

―

5 5
5.010
× 10
5.0 ×

105
5.05.0××10―—―—

7.6 × 10

1.0 × 1.0
10×6 106
3.0 × 3.0
10×6 10

—

—

―

6

—

69

7.9 × 102―

2.3 × 102

7.92.4× 102 106×

2.3 × 4.0102

2.4 × 106

4.0 × 105

1.5 × 10—

1.5 ×6 106

×6.06.0× 10
×6 1064.04.0× 106 106

× 105
―
―

—
6

10 × 106
82.0of× 2.0
16

Figure 5. The densities of growth-limiting bacteria detected from seawater (a): FLB, (b): PAB and four
macroalgal species (c): S. dupulicatum, (d): G. elegans, (e): C. ohkuboana and (f): U. pertusa collected atStn.2.
densitiesof growth-limiting
bacteria(●)
areexpressed
as
the
sum
ofPAB
algicidal
FigureFigure
4. The4.densitiesof Thegrowth-limiting
bacteria
detectedfrom seawater(a): FLB,
(b):
PAB
and
U.
The densities
densitiesofgrowth-limiting
bacteria
detectedfromseawater
(a):
FLB,
(b):
and
The
of
growth-limiting
bacteria
detectedfromseawater
(a):
FLB,
(b):
PAB
and
U. bacteria (AB:)
andgrowth-inhibiting
bacteria(GIB:
pertusa
biofilms(c):onpumicestone,(d):on
floating
cage
collected
atStn.1.
The
densitiesofgrowthU.
pertusa
biofilms
on pumicestone,(d):on floating
cage collected
atThe
Stn.1.
The densitiesof
pertusa
biofilms(c): (c):on pumicestone,(d): onfloating
cage collected
at Stn.1.
densitiesof growthlimiting
bacteria (●) bacteria
are expressedas the sumof sum
algicidal
bacteria(AB:(AB: ) and) and
growthgrowth-limiting(•) are expressedas theof algicidal
bacteria
growth-inhibiting
inhibiting
bacteria
The species
tested HAB
speciesand their abbreviations
bacteria(GIB:(GIB:). The tested).HAB
and theirabbreviations
are displayedare
at displayed
the bottom.atthe bottom.
). The tested HAB species and their abbreviations are displayed at the bottom.

3.3. Composition of Growth-Limiting Bacteria
The genus and class of GLB identified from different seawater fractions (FLB and PAB), and eachof the host macroalgae at both sites are shown in Figure 6. All of the GLB isolated from seawater andAppl. Sci. 2020, 10, x FOR PEER REVIEW

Appl. Sci. 2020, 10, x FOR PEER REVIEW

limiting bacteria (●) are expressed as the sum of algicidal bacteria (AB:) an
8 of 16
bacteria (GIB: ). The tested HAB species and their abbreviations are displayed

limiting bacteria (●) are expressed as the sum of algicidal bacteria (AB:) and growth-inhibiting
8 of 16
bacteria (GIB: ). The tested HAB species and their abbreviations are displayed at the bottom.

Appl. Sci. 2020, 10, 5658

8 of 16

Figure 5. The densities of growth-limiting bacteria detected from seawater (a): FL
macroalgal species (c): S. dupulicatum, (d): G. elegans, (e): C. ohkuboana and (f): U
Stn.2.detected
The densities
of growth-limiting
bacteria(●) fourare expressed as the sum of a
Figure5.5.Thebacteria
fromseawater
(a):
FLB,
(b):
and
Figure
Thedensities
densitiesofofgrowth-limiting
growth-limiting
bacteria
detectedfromseawater(a):FLB,(b):PABPAB
and four) and growth-inhibiting
bacteria(GIB:
detected from seawater (a):FLB, (b): PAB andU.
macroalgal
and (f):(f): U.
U. pertusa
pertusa collectedcollectedat
macroalgalspecies
species(c):(c):S.S.dupulicatum,
dupulicatum,(d):(d):G.
G.elegans,elegans, (e):(e): C.
C. ohkuboana
ohkuboana andng cage collected at Stn.1.atThe
densitiesof growthStn.2.
The
densitiesofgrowth-limiting
bacteria(•)
areexpressed
as
the
sum
of
algicidal
bacteria
Stn.2. The densities of growth-limiting bacteria (●) are expressed as the sum of algicidal bacteria(AB:
f algicidal bacteria (AB:(AB:) andgrowth) andgrowth-inhibiting
bacteria (GIB: ). The tested HAB species and their abbreviations are) and growth-inhibiting bacteria (GIB: ). The tested HAB species and their abbreviations are
B species and their abbreviationsatdisplayedare
at displayed
the bottom.
displayed at the bottom.
). The tested HAB species and their abbreviations are displayed at the bottom.

3.3. Composition of Growth-Limiting Bacteria
3.3. Composition of Growth-Limiting Bacteria
3.3. Composition of Growth-Limiting Bacteria
The genus and class of GLB identified from different seawater fractions (FLB and PAB), and eachand classGLB
identified
fromindifferent
seawaterfractions(FLB andfrom
PAB),and eachof the The
hostgenus
macroalgae
at of
both
sites
are shown
Figureand6.
All ofofthe
GLB
isolatedseawater
The genus
class
GLB
identified
from different
seawater fractions (
of
the
host
macroalgae
at
both
sites
are
shown
in
Figure
6.
All
of
the
GLB
isolatedfromseawaterandand macroalgal biofilms were comprisedof theAlphaproteobacteria,of thehostclasses
macroalgae
at both sites areGammaproteobacteria,
shown in Figure 6. All of the GLB isol
macroalgal
biofilms
werecomprised
theisolatedclassesfrom
Alphaproteobacteria,
andandFlavobacteria
at Stns.
1 and
2. Seven of
GLB
seawaterat Stn. Gammaproteobacteria,
1 wereclassified
themacroalgal
biofilms
were comprisedof theclassesasAlphaproteobacteria,
G
Flavobacteria
at
Stns.
1
and
2.
Seven
GLB
isolatedfromseawater
at
Stn.
1
were
classifiedasthegeneragenera of Nautella (Alphaproteobacteria) inFlavobacteria
FLB, Alteromonas,(Gammaproteobacteria)
at Stns.Pseudoalteromonas
1 and 2. Seven GLB
isolated from seawater at Stn. 1 wereof Nautella(Alphaproteobacteria)
in PAB
FLB, (Figure
Alteromonas,
Pseudoalteromonas(Gammaproteobacteria)
andandTenacibaculum(Flavobacteria) in
6a).(Alphaproteobacteria)In
total, five GLBinisolated
from U. pertusa
of Nautella
FLB, Alteromonas,
Pseudoalteromonas (GaTenacibaculum(Flavobacteria)
in PAB(Figure6a).
five
GLBfoundisolated
fromU.topertusagrown
grown
on the pumicestone andthe floating
cageInattotal,Stn.
1 wereto belongtheIn
generaofon GLB isolated fr
Tenacibaculum(Flavobacteria)
in PAB(Figure6a).
total, fivethepumice
stone
and
the
floating
cage
at
Stn.
1
were
found
to
belongtothegeneraofPseudoalteromonas, Vibrio (Gammaproteobacteria)
Aquimarina
respectively
the andpumicestone (Flavobacteria),
and the floating
cage at(Figure
Stn. 16b,c).
were found to bel
Pseudoalteromonas,
Vibrio(Gammaproteobacteria)
andWinogradskyella
Aquimarina (Flavobacteria),(FigureThe
eight GLB detected
in seawaterat Stn. Pseudoalteromonas,
2 were one(Flavobacteria)respectively
as FLB, andRoseobacter
Vibrio (Gammaproteobacteria)
Aquimarina (Flavobacteri6b,c).
The
eight
GLB
detectedinseawater
at
Stn.
2
were
one
Winogradskyella(Flavobacteria)as
FLB,(Alphaproteobacteria), Alteromonas, Pseudoalteromonas
Aquimarina,
6b,c). The andeightVibrio
GLB (Gammaproteobacteria)
detected in seawater andat Stn.
2 wereone Winogradskyell
Roseobacter(Alphaproteobacteria),
Alteromonas,
PseudoalteromonasandVibrio(Gammaproteobacteria)
andWinogradskyella (Flavobacteria) as PAB (Figure 6d). The twenty-seven GLB strains isolated from four
Aquimarina,
Winogradskyellaas from
PAB (Figure6d). The twenty-seven
GLB
strains
isolated
macroalgal
biofilms
consisted(Flavobacteria)of two genera
Alphaproteobacteria,
Phaeobacter,andRoseovarius,
from
four
macroalgal
biofilms
consistedoftwogenerafrom
Alphaproteobacteria,
Phaeobacter,andand three genera from Gammaproteobacteria, Alteromonas, Pseudoalteromonas, and Vibrio plus three genera

Appl. Sci. 2020, 10, x FOR PEER REVIEW

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Appl. Sci. 2020, 10, 5658

9 of 16

Roseovarius, and three genera from Gammaproteobacteria, Alteromonas, Pseudoalteromonas, and Vibrio
plus three genera of Flavobacteria, Aquimarina, Dokdonia and Winogradskyella (Figure 6e,f,g and h).
67% of GLB
found fromfour macroalgal
biofilms at(Figure
the natural
bed(Stn. 2)werethe bacteria
ofAbout
Flavobacteria,
Aquimarina,
Dokdonia
and Winogradskyella6e–h).
About
67%
of GLB
foundclassified
into the classbiofilms
Flavobacteria.
the2)only
GLB
from
all four into
macroalgae
fromfour macroalgal
at the Aquimarina
natural bedwas
(Stn.
were
thefoundbacteriaclassified
the classat
the natural bed(Stn. 2). was the only GLB found from all four macroalgae at the natural bed (Stn. 2).
Flavobacteria.
Aquimarina

Figure 6. The composition of growth-limiting bacteria (class and genus) isolated from seawater andthe different host macroalgae at Stn. 1 and 2. (Stn. 1: surface seawater (a), Ulva pertusa on pumice stone(b), U. pertusa on floating cage (c), Stn. 2: surface seawater (d), Sargassum dupulicatum (e), Gelidium
elegans
Cladophora
ohkuboana(g), U. pertusa (h)).Thein genus)
parenthesis
shows
the
numberandofFigure(f),6. Thecompositionof growth-limiting
bacteria number(class andisolatedfromseawaterstrains
used
for
16S
rRNA
gene
sequencing
for
identification.
the different host macroalgae at Stn. 1 and 2. (Stn. 1: surface seawater (a), Ulva pertusa on pumice stone(b), U. pertusa on floating cage (c), Stn. 2: surface seawater (d), Sargassum dupulicatum (e), Gelidium
elegans (f), Cladophora ohkuboana (g), U. pertusa (h)).The number in parenthesis shows the number ofstrains used for 16S rRNA gene sequencing for identification.

Appl. Sci. 2020, 10, 5658

10 of 16

4. Discussion
Recent findings of growth-limiting bacteria densely living in a biofilm of seagrass and macroalgae
have offered an insight into possible future HAB control by harnessing ecosystem services [51].
Meanwhile, the loss of seagrass and macroalgal beds due to human impacts have been accelerated overthe last half-century [52–54]. Such deterioration of coastal vegetation coincided with the increasing
occurrences of HAB [51,55]. The increasing toxic blooms of the dinoflagellate A. minutum accompaniedby the large-scale decline of seagrass beds around the Mediterranean coast is one of the specificcases [56]. Inaba et al. [37] documented that H. akashiwo cysts were found only from the sediments
where eelgrass had disappeared and studied sites where the abundant growth-limiting bacteria
detected rarely experienced blooms of paralytic shellfish toxin-producing dinoflagellate Alexandrium in
Puget Sound, WA, USA. On the positive side, the efforts to protect and restore seagrass and macroalgal
beds are underway worldwide [57,58]; for instance, vigorous restoration activities of seagrass bed in
Hinase, Okayama, Japan increased the natural bed more than twenty times since 1985 [57]. The efforts
will enhance the resistance of coastal areas against HAB events in the long run. However, this holistic
approach to prevent HABs may not be as applicable as direct controls, such as the use of flocculants [59].
Thus, it is crucial to develop an environmentally friendly measure that can be widely applicableand cost-effective.
Ulva can be attractive candidate organisms revealed to harbor GLB [34,37], growing fast comparedto other macroalgae or seagrass [60,61], relatively easy to culture [62], restoring water quality throughthe uptake of excess nutrients [63,64], releasing allelochemicals negatively affecting the growth of
HAB species [65,66], and having commercial importance in that they are frequently consumed under
the name “Aonori” in Asia [67,68]. Several Ulva species have already been tested to be organisms
composing large scale integrated multi-trophic aquaculture (IMTA) systems [67,69]. In the present
study, harmful algal growth-limiting bacteria (GLB) were found in the order of 106 CFU per 1 g ofU. pertusa grown on the pumice stone and the mobile floating cage at Stn. 1, showing the equivalentvalue to the natural growing macroalgal species at Stn. 2 as well as previous studies estimating thedensities of GLB on Ulva sp. and Gelidium sp. in Osaka Bay, Japan [34] and on U. lactuca in Puget
Sound, USA [37]. To our best knowledge, this is the first report to reveal that algal growth-limiting
bacteria can be harbored within a few months on the newly introduced Ulva thallus, suggesting thepossible future field application to control HABs by introducing Ulva beds as a source of GLB.
Previous studies have shown that a higher proportion of GLB was often associated withparticles [28,44]. In the study in Puget Sound, most isolated bacteria from seawater having more
extensive activity ranges were in the particle-associated form [37]. In the present study, the density of
PAB (Particle-associated bacteria) as GLB was higher than FLB (Free-living bacteria) at the natural
macroalgal bed (Stn. 2), killing most of the tested HAB species except A. catenella (Figure 5b). Moreover,
88% of GLB detected from seawater at the natural macroalgal bed was the same bacterial genera as GLB
detected from macroalgal biofilms (Figure 6). It is known that macroalgal beds are one of the essential
sources releasing particulate and dissolved organic matter (POM and DOM) to the surrounding
environments [70–72]. The POM derived from the macroalgae at Stn. 2 may have increased substrateavailability for specific bacterial communities with high exoenzymatic activities [73–75] that resulted
in frequent detection of GLB as PAB forms in the water. This could be reasonably explained by highly
dense GLB harbored on a macroalgal surface being provided synchronously with macroalgal-derived
POM to the surrounding water.
Many bacteria previously reported as algicidal and/or growth-inhibiting bacteria were among
the Cytophaga/Flavobacterium/Bacteroidetes (CFB) group [27]. They are also critical players for the
degradation of organic matter in aquatic environments [76]. Ecologically important roles toward
macroalgae of the CFB group have been demonstrated, such as controlling morphogenesis [77,78],
promotion and/or inhibition of spore germination, and colonization [79,80], close association withdiseases [81], etc. In the present study, more than half of GLB were identified as bacteria belonging
to the class Flavobacteria. Notably, the genus Aquimarina within the class Flavobacteria were foundAppl. Sci. 2020, 10, 5658

11 of 16

Appl. Sci. 2020, 10, x FOR PEER REVIEW

11 of 16from all of the macroalgae and 25% of GLB in seawater samples at Stn. 2 (Figure 6). With regard to
the growth-limiting
activity
range,
all isolated
GLB isolated
from seawater
andsamples
Ulva samples
Stn. 1foundweregrowth-limiting
activity
range,
all GLB
from seawaterand Ulva
at Stn. at
1 werefound
to exhibit
specific
activitiesin 92%
that 92%of GLBnegatively
affected
growthofofonly
onlyoneoneHAB
HAB
toexhibit
specific
activities
in thatof GLBnegatively
affectedthethegrowthspecies(C.
antiquaorH.
circularisquama)(Figure7a);
however,
GLB
isolatedfromnaturalmacroalgae
species (C. antiqua or H. circularisquama) (Figure 7a); however, GLB isolated from natural macroalgae
bed showed
showed aa much
much broader
broader range
range ofof activities
activities onon tested
tested HAB
HAB species
species (Figure(Figure 7b).
7b). About
About 90%
90% ofof GLB
GLB
bed
affecting thethe growth
growth ofof moremore than
than oneone HAB
HAB species
species were
were found
found toto be
be Flavobacteria,
Flavobacteria, which
which consisted
consistedaffectingof
Aquimarina
andDokdonia(Figure7b).
Aquimarina
isolated
from
red
alga
on
the
coast
of
China isisof Aquimarina and Dokdonia (Figure 7b). Aquimarina isolated from red alga on the coast of China
shown toto exhibit
exhibit diverse
diverse agarase
agarase activities,
activities, enhancing
enhancing thethe degradation
degradation ofof hosthost algal
algal tissue
tissue [82].
[82]. These
These
shown
resultssuggestthemultiple
roles
of
the
membersof
Flavobacteria
diffusingaroundmacroalgal
beds.
results suggest the multiple roles of the members of Flavobacteria diffusing around macroalgal beds.
Thephysiologicalandbiochemical
characteristics
ofmacroalgae
predetermine
thecompositionofThe physiological and biochemical characteristics of macroalgae predetermine the composition of
their biofilm[83],
andandthe host
species-specificmicrobial
association
in aquatictheirbiofilm microbial
microbialcommunities
communities[83],
the host
species-specificmicrobial
associationinangiosperms
is
known
[84].
It
is
worth
notingthatnaturalmacroalgal
beds
with
higheraquatic angiosperms is known [84]. It is worth noting that natural macroalgal beds withdiversity
highermay offermay
varioussubstratessubstrates
for diversefor
GLB,
functioning
effectivelyeffectively
as bloom control
for
diversityofferinhabiting
various inhabiting
diverse
GLB, functioning
as bloommultiplealgal
species(Figure7b).(Figure 7b).
controlforharmful
multiple
harmfulalgal
speciesFigureFigure7.
7.The
Thegrowth-limiting
growth-limitingactivity
activityranges
rangesofofGLB
GLBagainst
againstall
allfive
fiveharmful
harmfulalgal
algalspecies
species(A.
(A.catenella,catenella,C.
, andand K.
K. mikimotoi)
mikimotoi) isolated
isolated fromfrom (a)(a) Stn.
Stn. 11 andStn. 2.
C. antiqua,
antiqua, H.
H. akashiwo,
akashiwo, H.
H. circularisquama
circularisquama,and (b)(b) Stn.
2. It
Itincludesall GLB
GLBstrains
strainsisolated
isolatedfrom
fromseawatermacroalgal
samples
at eachsite. Total
includes all
seawaterandandmacroalgal
samples
at eachsite. Total
strainsstrainstestedtestedare shownin parenthesisin the lowerof thepie chart.
are shownin parenthesisin the lower
center centerof the pie
chart.

5. Conclusions
Conclusions
5.
The present
present study
study illustrated
illustrated thatthat GLB
GLB withwith equivalentequivalent value
value asas naturally
naturally growing
growing macroalgae
macroalgae
The
could
be
harboredonthesurfaceofartificiallyintroducedU.
pertusa
for
less
than
three
months,could be harbored on the surface of artificially introduced U. pertusa for less than three months,
providingnovel
insightintothepotential
use
of
the
artificial
introductionofU.
pertusabeds asasproviding novel insight into the potential use of the artificial introduction of U. pertusa beds
biological control
control ofof HABs.
HABs. Results
Resultsfrom
fromthethenatural
naturalmacroalgal
macroalgalbed
bedsuggested
suggestedthat
thathigher
highermacroalgal
macroalgal
biologicaldiversity
providediverseinhabiting
substratesfor
GLB,
functioning
effectivelyasbloom
controlfor
for
diversity provide diverse inhabiting substrates for GLB, functioning effectively as bloom control
multiple harmful
harmful algal
algal species.
species. ItItshould
shouldbe
be also
also emphasizedemphasized thatthat thethe protection
protection andand restoration
restoration ofofmultiplenaturalmacroalgal
beds
may
serve
as
not
only
criticalhabitatsfornumerous
marineandestuary
natural macroalgal beds may serve as not only critical habitats for numerous marine and estuaryorganisms but
but also
also one
one ofof thethe original
original hotspots
hotspots ofof GLB,
GLB, contributing
contributing toto mitigating
mitigating HABs
HABs inin coastal
coastalorganisms
environments. Further
Furtherinvestigations
investigationstotounderstandunderstandthe
thediffusive
diffusivebehavior
behaviorofofGLB
GLB areare neededneeded totoenvironments.
achievethepractical
application.
achieve the practical application.
Author Contributions: All authors contributed the project. N.I. wrote the manuscript. I.K. supported the
Author Contributions: All authors contributed the project. N.I. wrote the manuscript. I.K. supported thesampling and performed the algal and part of the molecular analyses. S.N. supported molecular analyses. T.S.
sampling
performed
the algal preparation.
and part of themolecular
analyses.
S.N. supported
molecular
T.S.
supportedandsamplingand zoospore
N.I.,
K.M., A.Y.
and I.I. conceived
and designed
theanalyses.
experiments.
supported
samplingand zoospore
preparation.
N.I., K.M.,
A.Y.
and I.I. conceived
andhavedesignedthe experiments.
All co-authors
participatedin discussions
and reviewof themanuscript.
All authors
read andagreed to the
publishedversionof the manuscript.
Allco-authors
participatedin discussions and review of the manuscript. All authors have read and agreed tothepublishedversionofthemanuscript.
Funding: This study was supported
financially in part by a Grant-in-Aid for Scientific Research grants 18K14506(Early Career Scientists) from the Japanese Society for the Promotion of Science (JSPS) and a Grant-in-Aid
Funding: This study was supported financially in part by a Grant-in-Aid for Scientific Research grants 18K14506(Early Career Scientists) from the Japanese Society for the Promotion of Science (JSPS) and a Grant-in-Aid(Technological developments for characterization of harmful plankton in the seawater: No. 16808839) from the
Ministry of Agriculture, Forestry and Fisheries of Japan.

Appl. Sci. 2020, 10, 5658

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(Technological developments for characterization of harmful plankton in the seawater: No. 16808839) from the
Ministry of Agriculture, Forestry and Fisheries of Japan.
Acknowledgments: We would like to show sincere thanks to the members of Fisheries Engineering Research
Team, Civil Engineering Research Institute for Cold Region (CERI), Japan for administrative support, especially,
Sawako Shirai, technical assistant. We also express our gratitude to Masaru Okamoto, Shinkotec co., ltd for thesuggestions on pumice stone.
Conflicts of Interest: The authors declare no conflict of interest.

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3.
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10.
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12.
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14.
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16.

17.
18.

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