TYPE Original Research
PUBLISHED 15 September 2022
DOI 10.3389/fmars.2022.998711

OPEN ACCESSEDITED BY

Ondrej Prasil,
Institute of Microbiology, Academy of
Sciences of the Czech Republic(ASCR), Czechia
REVIEWED BY

Bernard Lepetit,
University of Konstanz, Germany
Douglas Andrew Campbell,
Mount Allison University, Canada
Ane Kvernvik,
The University Centre in Svalbard,
Norway*CORRESPONDENCE

Yuri Fukai
fukai@ees.hokudai.ac.jp
Koji Suzuki
kojis@ees.hokudai.ac.jp
SPECIALTY SECTION

This article was submitted to
Aquatic Microbiology,
a section of the journal
Frontiers in Marine Science
RECEIVED 20 July 2022
ACCEPTED 29 August 2022
PUBLISHED 15 September 2022
CITATION

Fukai Y, Matsuno K, Fujiwara A andSuzuki K (2022) Photophysiologicalresponse of diatoms in surface
sediments to light exposure: A
laboratory experiment on adiatom community in sedimentsfrom the Chukchi Sea.
Front. Mar. Sci. 9:998711.
doi: 10.3389/fmars.2022.998711
COPYRIGHT© 2022 Fukai, Matsuno, Fujiwara andSuzuki. This is an open-access article
distributed under the terms of theCreative Commons Attribution License(CC BY). The use, distribution or
reproduction in other forums ispermitted, provided the original
author(s) and the copyright owner(s)are credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms.

Photophysiological response ofdiatoms in surface sediments tolight exposure: A laboratory
experiment on a diatom
community in sediments fromthe Chukchi Sea
Yuri Fukai 1*, Kohei Matsuno 2,3, Amane Fujiwara 4
and Koji Suzuki 1,5*
1Graduate School of Environmental Science, Hokkaido University, Sapporo, Japan, 2 Faculty/
Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Japan, 3 Arctic ResearchCenter, Hokkaido University, Sapporo, Japan, 4 Research Institute for Global Change, Japan Agency
for Marine-Earth Science and Technology, Yokosuka, Japan, 5 Faculty of Environmental Earth
Science, Hokkaido University, Sapporo, Japan

Diatoms form dense blooms in the Paciﬁc Arctic region from spring to summer,
supporting the unique benthic-pelagic coupling ecosystems. Although the
Arctic has a severe light-limited season from autumn to winter, diatoms canproliferate in spring when sufﬁcient light becomes available for photosynthesis.
One of the crucial strategies for diatoms to survive in unfavorable growing
conditions is to form resting stages. Because of enhanced primary and export
production in the Paciﬁc Arctic shelves, many viable diatom resting stages canbe detected in the surface sediments. However, little is known about thephotophysiological response of viable diatom cells, including resting stages, insediments to light availability. We conducted a laboratory experiment
investigating the photophysiological capabilities of the diatom cells
containing resting stages using surface sediments from the Chukchi Sea
shelf. As a result, diatoms grew dramatically after light exposure, andChaetoceros socialis complex highly contributed to the enhanced diatom
abundance. Their photophysiological changes were also evident from the
maximum quantum efﬁciency (Fv/Fm) of photochemistry in photosystem II,
13
C-based photosynthetic-energy (PE) parameters, diadinoxanthin (DD)diatoxanthin (DT) pool size, and the de-epoxidation state (DES) of DD. Even
after the excess light exposure suppressed the photosynthetic activity in the
microalgal cells, the diatoms recovered quickly, indicating the highphotophysiological plasticity to dynamic light changes. Therefore, our results
suggest that diatoms in surface sediments have a high seeding potential for
blooms in the Paciﬁc Arctic shelf region.
KEYWORDS

diatom, diatom resting stages, photophysiology, sediment, Paciﬁc Arctic shelves,
Chukchi Sea

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contributions of diatom resting stages in sediments to springdiatom blooms have been reported in temperate Hakata Bay,
Japan (Shikata et al., 2009) and the coastal shallow Arctic
Svalbard (Hegseth et al., 2019). Previous studies observed
increases in cell abundance, biomass, and pigment
concentration in seawater from the sediments (e.g., Shikata
et al., 2009), but there is little evidence of how algal
assemblages respond to light regimes when they germinate
and resume photosynthesis. It is critical to pile up thephysiological knowledge about the carbon ﬁxation by diatoms
in sediments because they can play a signiﬁcant role in primary
production in the Arctic Ocean after a long dark period (Hegseth
et al., 2019; Johnsen et al., 2020). In the Paciﬁc Arctic shelves,
sediments can be re-suspended by the fast ﬂow in the Bering
Strait (Abe et al., 2019) and strong wind events (Uchimiya et al.,2016). In addition, irradiance reaching the shallow seaﬂoor of
the Paciﬁc Arctic may increase because of the future thinner sea
ice and earlier sea ice retreat (Nicolaus et al., 2012; Horvat et al.,2017). Therefore, our understanding of photophysiologicalresponses by microalgae in sediments to light exposure would
provide valuable insights into the primary production processes
in the Paciﬁc Arctic shelves.
Natural diatoms face considerable variation in irradiance,
which is especially prominent in the Arctic (Cohen et al., 2020).
For acclimating to the light variation, diatoms possess
photophysiological strategies. Short-term (minutes to hours)
acclimation in diatoms mainly includes the diadinoxanthindiatoxanthin cycle (i.e., de-epoxidation of diadinoxanthin to
diatoxanthin) and non-photochemical quenching (NPQ) thatdissipate excess excitation energy and quench chlorophyll
ﬂuorescence (Goss and Lepetit, 2015; Yan et al., 2019).
Additionally, changes in the structure and composition of thephotosystems in diatoms support the hourly to daily
photoacclimation (Brunet et al., 2011). In hourly to daily highlight conditions, changes in cellular pigment composition can beobserved, and photoprotective carotenes are generally increased intheir cells (Brunet et al., 2011). In contrast, the contents of lightharvesting photosynthetic pigments can be decreased
concomitantly (Brunet et al., 2011). To explore thephotosynthetic response of microalgae, including diatoms, tolight exposure, the photosynthesis-energy (P-E) curve
experiment has been conducted (e.g., Isada et al., 2019; Yoshida
et al., 2020b). From the P-E relationship, the maximum carbon
ﬁxation rate (P m a x ), light utilization coefﬁcient (a),photoinhibition parameter (b), and photoacclimation index (Ek)can be derived to estimate the photoacclimation status of
microalgae (Sakshaug et al., 1997). Although photophysiologicalparameters, including P-E parameters, were assessed in manystudies for microalgal communities (e.g., Isada et al., 2019;
Yoshida et al., 2020b), there have been few studies focused on
photophysiological responses by microalgae in sediments.
In this study, we aimed to unravel the responses ofdiatoms that survived in the prolonged darkness of ca. 9

1 Introduction
The Paciﬁc Arctic region with seasonal sea ice is experiencing
rapid seasonal sea ice retreat, along with other environmental
changes and biological responses in lower to upper trophic
organisms (Grebmeier and Maslowski, 2014). The northern
Bering and Chukchi Seas within the Paciﬁc Arctic region haveshallow shelves and display large microalgal blooms from spring tosummer, with the highest daily primary production among theworld’s oceans (Springer et al., 1996). The algal blooms are
traditionally thought to initiate from the proliferation of ice algaein the marginal ice zone, followed by phytoplankton blooms inopen waters (e.g., Horner and Schrader, 1982). Recently, a massive
algal bloom occurring under the sea ice, the so-called “under-ice
phytoplankton bloom” (Arrigo et al., 2012), has been widely
recognized on the pan-Arctic scale (Ardyna et al., 2020). On theother hand, for proliferation, the microalgae need to survive in the
prolonged darkness (up to several months annually) during winterusing various strategies (McMinn and Martin, 2013).
Diatoms generally dominate algal communities in sea ice,water column, and surface sediments of the Paciﬁc Arctic region(e.g., von Quillfeldt, 2000; Sergeeva et al., 2010; Ardyna et al.,2020; Fukai et al., 2021; Suzuki et al., 2021). Diatoms form
resting stages under unfavorable conditions for their growth,such as macronutrient limitation (Durbin, 1978; Garrison, 1984;
Smetacek, 1985; McQuoid and Hobson, 1996), iron starvation(Sugie and Kuma, 2008), and low-light conditions (Hargraves
and French, 1983), so that they can survive even in theunsuitable environments. Because most diatom resting stages
have heavy frustules, they can quickly sink to the seaﬂoor andsometimes highly contribute to the sinking particulate organiccarbon (POC) ﬂux (Rynearson et al., 2013). The sinking POC
ﬂux in the northern Bering and Chukchi Seas, supported bydiatoms, sometimes reaches the highest ever documented acrossthe global oceans (Lalande et al., 2020; O’Daly et al., 2020).
Diatom cells settled on the seaﬂoor support the highconcentration of diatom resting stages in sediments of thenorthern Bering and Chukchi Seas (Fukai et al., 2021).
Therefore, the distribution of diatom resting stage assemblages
in sediments might reﬂect the past blooms in the water column(Pitcher, 1990; Itakura et al., 1997). The diatom assemblages in
sediments of the Paciﬁc Arctic region could also be inﬂuenced by
the timing of sea ice retreat and the amount of primary
production in the water column (Fukai et al., 2019; Fukai
et al., 2021). The open-water bloom-forming diatom species
such as Chaetoceros spp. and Thalassiosira spp. observed in thewater column (von Quillfeldt, 2000; Sergeeva et al., 2010; Fukai
et al., 2020; Suzuki et al., 2021) are mainly dominated in theresting stage community in the sediments of the northern Beringand Chukchi Seas (Fukai et al., 2021).
The diatom resting stages sinking to and accumulated on theseaﬂoor can germinate and resume cell growth when they
assimilate sufﬁcient light (Hollibaugh et al., 1981). The highFrontiers in Marine Science02

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10.3389/fmars.2022.998711

months after sampling from surface Chukchi shelf
sediments to light exposure with particular reference totheir photosynthetic physiology in terms of pigmentcomposition, the maximum quantum yield ofphotochemistry (F v /F m ) in photosystem II, and theP-E parameters.

2.1.2 Quantiﬁcation of viable diatom cells in
sediments
The concentrations of viable diatom cells in the sediment
samples were analyzed by the most probable number (MPN)
method (Imai et al., 1984; Imai et al., 1990). The sediments
stored for four months in the dark were homogenized andsuspended in the sterilized ﬁltered seawater at a concentration of
0.1 g mL−1 (=100 dilution). The subsequent serial tenfold dilutions(10−1 to 10−6) were made with f/2 medium (Guillard and Ryther,
1962). Then 1 mL aliquots of the diluted suspensions were
inoculated into ﬁve replicate wells of disposable tissue culture
plates (48 wells). Incubation was carried out at a temperature of
5°C and under the white light-emitting diode (LED) light (LXHLBW02, Lumileds, Netherlands) with about 50 mmol photons m−2
s−1 and a 12 h light:12 h dark photocycle for 10 days, following
Fukai et al. (2021). The appearance of vegetative cells of planktonic
diatoms in each well (i.e., “positive well”) was examined using an
inverted light microscope. Based on the number of positive wells
out of the ﬁve replicates in each dilution series, the most probable
number (MPN for a series of 5 tenfold dilutions) of each diatom
species in the sediment sample (MPN cells g−1 wet sediment) was
then calculated according to the statistical table byThrondsen (1978).

2 Materials and methods
2.1 Spatial distribution of viable diatom
assemblages in sediments
2.1.1 Sampling of sediments
Sampling was conducted on the shallow shelves in thesouthern Chukchi Sea (Figure 1) from October 7–20, 2020,
during the R/V Mirai MR20-05C expedition (Japan Agency
for Marine-Earth Science and Technology). Sediment samples
were collected using a Smith-McIntyre bottom sampler, andthen the surface sediments (0–3 cm) were divided using anacrylic tube and a spatula. Subsamples of the surface sediments
were stored in darkness at 3°C until further experimental
procedures on land.

FIGURE 1

Locations of the sediment sampling in October 2020 and the horizontal distribution and composition of viable diatom cells in the Chukchi Sea
sediments as estimated by the MPN method. Color contours and numbers indicate the bottom depth and station name, respectively.

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2.2.3 Microalgal pigments

2.2 Laboratory experiment forinvestigating benthic algal
photophysiology

Samplings for microalgal pigments were conducted everyday (i.e., from day 0 to day 7). Samples of 10 or 20 mL wereﬁltered onto Whatman GF/F ﬁlters under a gentle vacuum(<0.013MPa). The ﬁlter was immediately frozen in the deep
freezer at −80°C until further analysis. Microalgal pigments wereextracted using the N, N-dimethylformamide (DMF) beadbeating technique and analyzed by Ultra-High Performance
Liquid Chromatography (UHPLC) following Suzuki
et al. (2015).
The chlorophyll a (chl a) speciﬁc growth rate (mchl a, day-1)
was calculated as the slope of the regression line plotting thenatural log-transformed chl a concentration against the
incubation time. The concentrations of light-harvesting
photosynthetic pigments (LHP) were estimated with the sumof chl a, chl c, and fucoxanthin, whereas the sum of
diadinoxanthin (DD) and diatoxanthin (DT) was treated as anindicator of photoprotective pigments. The size of xanthophyll
pool (DD-DT pool) was assessed as the sum of DD and DT
normalized by chl a (i.e., ([DD] + [DT])/[chl a]) (Yoshida et al.,2020a), where [DD], [DT], and [chl a] indicate theconcentrations of diadinoxanthin, diatoxanthin, and chl a,respectively. In addition, the pool size of carotenes wasestimated from the concentration of carotenes normalized bychl a (i.e., [carotenes]/[chl a]), where [carotenes] denotes theconcentration of carotenes.
The de-epoxidation state index (DES; Ruban et al., 2004) inthe diadinoxanthin-diatoxanthin cycle was calculated as follows:

2.2.1 Viable diatom cells in sediments
To accurately determine the concentrations andcomposition of viable diatom cells in sediments used in thelaboratory experiment, the MPN method was conducted again
immediately before the laboratory experiment (Imai et al., 1984;
Imai et al., 1990) because the experiment was implemented after
5 months from the estimation of the spatial distribution of viable
diatoms (see section 2.1.2). The procedures were the same,
except that the incubation was carried out at 3°C and under
the white ﬂuorescent light of about 60 mmol photons m−2 s−1
with a 12 h light:12 h dark photocycle for 9 days.

2.2.2 Experimental set-up
Sediments and the near-bottom seawater collected at station
35 (bottom depth: 53.9 m) and a site (69.03°N, 168.83°W)(bottom depth: 51.3 m) near station 7, respectively, were used
for the experiment. Sediments were sampled as described above.
Subsamples of the surface sediments were stored in darkness at
3°C for about 9 months until the experiment was started. The
near-bottom seawater was collected with a Niskin water sampler,ﬁltered immediately using a Merck Sterivex ﬁlter (pore size 0.22mm), and sterilized by an autoclave (121°C, 20 minutes). The
sterilized ﬁltered seawater was stored at 3°C until use.
For the sediment incubation, a sediment subsample (1.1 g) wassuspended into the 4.4 L sterilized ﬁltered seawater. Then, thesediment suspended seawater was stored in darkness at 3°C forabout 16 hours to acclimate to the experiment temperature. Afterthe acclimation, samplings and sample analyses (see below for
details) on day 0 were conducted before the sediment suspended
seawater was divided into two 2 L bottles, which were moved into
the experimental incubator at 3°C with white ﬂuorescent light(LMGA1WG21K50A410F, Prince Industry Inc., Japan) of 300
mmol photons m−2 s−1 (High Light; HL) or 30 mmol photons m−2
s−1 (Low Light; LL), respectively. The two irradiance levels were set
to simulate different light regimes in the water column. A squareshaped illumination cycle was set at 12 h light: 12 h dark for 7 daysin both light conditions. The experiments were conducted threetimes to test the repeatability of the results.
In this study, the following parameters were determined: theconcentrations of viable diatom cells, including resting stages, insediments, the composition and concentrations of microalgal
pigments, the maximum photochemical quantum yield (Fv/Fm)of photosystem II for microalgae, the concentrations of diatom
cells, and 13 C based P-E parameters. Samples for theseparameters, except for the concentrations of viable diatom
cells in sediments, were obtained from HL and LL bottles
according to the procedures detailed below.

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DES = ½DT =ð½DD + ½DT Þ
When the concentrations of DD and DT were null, the data
were excluded from the calculation.

2.2.4 Maximum photochemical quantum yieldof photosystem II
The maximum quantum yield of photochemistry inphotosystem II (i.e., Fv/Fm) for microalgae was measured every
day using a pulse amplitude modulated ﬂuorometer (WaterPAM, Walz, Germany). Samples were acclimated in darkness at
3°C for about 1 hour before the measurement and then put into aquartz cuvette. The minimum ﬂuorescence (Fo) after the darkacclimation and the maximum ﬂuorescence under the saturation
pulse (Fm) were determined. The saturating pulse was provided
by red light-emitting diodes (LEDs), which had a peakillumination at 660 nm of 4000 mmol photons m−2 s−1 andlasted for 600 ms. The Fv/Fm was calculated as follows:
Fv =Fm = ðFm − Fo Þ=Fm
The ﬂuorescence measurements were conducted twice
per sample.

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10.3389/fmars.2022.998711“phytotools” package (Silsbe and Malkin, 2015) of R software(version 4.0.4, R Development Core Team, 2021: https://www.rproject.org).

2.2.5 Diatom cell counting
Samples for diatom cell counting were obtained every day.
The sample of 9 mL was ﬁxed by neutral formalin at a ﬁnal
concentration of 2%. If necessary, the samples were then settled
and concentrated by 2.0- to 3.3- fold. Aliquots (1 mL) of the subsamples were transferred to a glass slide to count and identify thediatoms under an inverted microscope (Olympus BH2 BHS
system, Olympus, Tokyo, Japan; 10 × oculars and 40 × objective
lenses). The diatoms were counted and identiﬁed from morethan 150 cells, including the sufﬁcient counting (more than 50
cells) of the dominant species (Edler and Elbrächter, 2010). It
was difﬁcult to distinguish the resting stages of the genus
Thalassiosira from their vegetative cells; therefore, the total cellnumbers were estimated in this study. The cell-speciﬁc growthrate (mcell, day-1) was calculated as the slope of the regression line
plotting the natural log-transformed cell concentrations against
the incubation time.



B





exp −bPAR PBEPAR inc

s

B



s

where the superscript B denotes the normalization by chl aconcentration, and P is the photosynthetic rate, EPAR inc is theirradiance at each position of the incubator, P s is the
hypothetical maximum photosynthetic rate in the absence ofphotoinhibition, a is the initial slope of the P-E curve, and b is
the photoinhibition parameter. The maximum photosynthetic
rate, PBmax, is calculated as follows:

B
= PsB
Pmax

aB
B
a + bBbB
Ba + bBb B =a B

The photoacclimation index, Ek, was derived from thefollowing equation.

2.2.6 13C-based Photosynthesis-Energy curve
experimentB
Ek = Pmax=a B

The Photosynthesis-Energy (P-E) curve experiments wereconducted on day 0, 1, 4, and 7. The culture samples weredispensed into eleven 30 mL acid-cleaned polycarbonate bottles.
As an initial sample, a bottle of a sample without any isotope
addition was ﬁltered onto a Whatman GF/F glass ﬁber ﬁlter precombusted over 5 hours at 450°C. The other ten sample bottles
were inoculated with a known amount of NaH13CO3 solution(99 atom% 13C, Shoko Co., Ltd.), equivalent to ca. 10% of
dissolved inorganic carbon (DIC) in the medium. These 13Cenriched samples were set on an incubator by Babin et al. (1994)
and kept at 3°C under ten different light intensities from 1.47 to1770 mmol photons m−2 s−1 using a PCS-UMX250 light source(Nippon PI Co., Ltd.) for 2 hours. After incubation, the samples
were ﬁltered on pre-combusted GF/F glass ﬁber ﬁlters. All ﬁlters
were stored in a deep freezer at −80°C until further analysis. The
duplicate samples of DIC in the medium without NaH13CO3
enrichment were obtained in 120 mL glass bottles. The DIC
concentrations were determined using a Total Alkalinity
Titrator ATT-05 (Kimoto Electric Co., Ltd) with authentic
standards (KANSO Technos).
The ﬁlters were thawed, exposed to the fumes of HCl toremove inorganic carbon, and entirely dried in a desiccator
under a vacuum. The amount of particulate organic carbon(POC) and the atom percent of 13C in each sample were
determined by an online element analyzer (FlashEA1112,
Thermo Finnigan)/isotope ratio mass spectrometer (Delta-V,
Thermo Finnigan).
The chl a concentration normalized photosynthetic carbon
ﬁxation rates were calculated following Hama et al. (1983) basedon the 13C technique and chl a concentration measured by
UHPLC. The P-E curve ﬁtting and parameters were obtained
from the photoinhibition model of Platt et al. (1980) with the

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PB = PsB 1 − exp −aPAR PBEPAR inc

To estimate the maximum quantum yield of carbon ﬁxation,
microalgal absorption coefﬁcient values under the HL and LL
conditions were measured on day 7. The duplicate water samples(10 or 20 mL) were ﬁltered onto Whatman GF/F ﬁlters. The
ﬁlters were covered with aluminum foil and stored in a deep
freezer (−80°C) until further analyses. A spectrophotometer(MPS-2450, Shimadzu) equipped with an end-on-type
photomultiplier tube was used to measure the absorption
spectra of the total particles on the ﬁlters at every 1 nm. Then,the algal pigments on the ﬁlters were extracted by soaking inmethanol, and the absorption spectra of detritus on the ﬁlter
were measured in the same manner. The measured absorption
spectra of the total particles and the detritus were corrected forthe path-length ampliﬁcation effect following the equation ofStramski et al. (2015).
ODs ðl Þ = 0:679

1:2804
ODf ðl Þ

where ODs (l) and ODf (l) are the absorption spectra ofsuspension and ﬁlter, respectively. Then, the absorption
coefﬁcient (ap (l)) values were calculated for the total particles
and the detritus, respectively, following (Kishino et al., 1985):
ap ðl Þ = 2:3

ODs ðl Þ

s=v

where s is the ﬁltered area (m2) and v is the ﬁltered volume (m3)of the samples. The absorption coefﬁcient of microalgae (aph)was obtained by subtracting the absorption coefﬁcient of the
detritus from that of the total particles.
B
The P-E parameters for light absorbed by microalgae (aPUR
B
and bPUR ) were derived from the equations described above
using the photosynthetic absorbed radiation at each position of
the incubator (EPUR inc). The subscript PUR indicates the light05

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and 29% of the diatoms at stations 30 and 31, respectively. The
other centric diatoms, including Actinoptychus sp., Paralia
sulcata, Skeletonema spp., Leptocylindrus spp., and Odontellaspp., appeared from the sediments but little contributed to thediatom communities. Pennate diatoms, such as Asteroplanus
karianus, Fragilariopsis spp., Navicula spp., and Cylindrothecaclosterium, emerged from the sediments at all stations, but the
proportion was relatively low over the study region (0.47–13%).

absorbed by microalgae, and the EPUR inc was calculated asfollows (Dubinsky, 1980):
Z 700

EPAR inc ðl Þaph ðl Þdl

E ðl Þ
EPAR inc ðl Þ = Z 700 L
EL ðl ÞdlEPAR inc

EPUR inc =

400

where

400

3.2 Incubation experiment

where EL (l) is the incubator lamp spectrum. The maximum
quantum yield of carbon ﬁxation (Fc max, mol C (mol photon)-1)
was calculated by the following equation:
fc max = 0:0231½chl a

3.2.1 Viable diatoms in sediments
The concentrations of viable diatoms in sediments at station 35,
estimated immediately before the onset of the experiment, were
6.0 × 105 ± 4.7 × 105 MPN cells g−1 wet sediment. The total
concentrations decreased from that of the former estimation of
viable diatom cells at station 35 (t-test, p< 0.05), while the diatom
composition and the appearing diatom species were not different
from each other (Figures 1, 2 and Supplementary Table 2). Centric
diatoms accounted for 97%, and the genera of Chaetoceros andThalassiosira contributed to 93% of the community. Chaetocerossocialis complex was primarily dominated (79%). The other centric
diatoms, including Attheya spp., Detonula pumila, Odontella spp.,
P. sulcata, Skeletonema spp., L. danicus, and Melosira sp.,sometimes appeared from the sediment but little contributed tothe community. Pennate diatoms, such as A. karianus,
Fragilariopsis spp., and Navicula spp., also appeared, but therelative abundance was very low.

B
aPUR

where [chl a] is the chl a concentration (mg m-3) and 0.0231 is afactor to convert milligrams of carbon to moles, mmol photons to
mol photons, and hours to seconds (Isada et al., 2009).

2.3 Statistical analyses
Differences among treatments and incubation time or period
were tested using one-way analysis of variance (ANOVA) andpost hoc Tukey-Kramer test. To compare differences in thevalues between day 0 and day 1 in HL or between HL and LL,
t-test was conducted for each treatment. Also, the differences in
the concentration of viable diatom cells in the sediment at
station 35 were estimated twice using the MPN method witht-test. All statistical analyses were performed using R software(version 4.0.4, R Development Core Team, 2021).

3.2.2 Microalgal pigments
The sum of chl a and chl c concentration increased in both
HL and LL and became more than double the initial level in HL
on day 5 and in LL on day 4 (Figures 3A, B, and Supplementary
Table 3). Similarly, fucoxanthin concentrations increased inboth treatments (Figures 3A, B, and Supplementary Table 3).
In HL on day 4 and LL on day 3, the concentrations offucoxanthin reached double the initial levels.
The trends of the concentration of diadinoxanthin anddiatoxanthin (i.e., [DD] + [DT]) were also distinct between
HL and LL (Figures 3C, D, and Supplementary Table 3). In HL,
the [DD] + [DT] initially decreased, reduced by 22% of initial on
day 1, and thereafter increased after day 3 (Figure 3C). On theother hand, the [DD] + [DT] gradually increased with time
under LL (Figure 3D). The LHP concentrations under HL and
LL increased from day 3 and day 2, respectively, and reachedabout 10 times on day 7 from the initial value (Figures 3C, D). In
HL, the DD-DT pool size estimated from [DD] + [DT])/[chl a](Figure 3E) decreased on day 1 due to the decrease in [DD] +[DT], although the decline was not signiﬁcant (t-test, p =0.05).
After day 1, the DD-DT pool size under HL returned to theinitial level by day 3, whereas the ratio in LL decreased to almost

3 Results
3.1 Spatial distribution of viable diatom
cells in sediments of the Chukchi
Sea shelf
The viable diatom cells determined by the MPN method
ranged from 1.90 × 105 to 1.91 × 106 MPN cells g−1 wet sediment(Figure 1 and Supplementary Table 1). The concentrations
reached 106 MPN cells g−1 wet sediment only at station 35.
Centric diatoms were predominant throughout the region, andthe relatively high contributions of Chaetoceros socialis complex
to the total viable diatom cells were found at almost all stations(40–89%), except stations 9 and 29 (31% and 18%, respectively).
At stations 9 and 29, Attheya spp. were dominant in the viable
diatoms, while the genus comprised only 0.30–21% at the otherstations. Other genera showed a locally high proportion. For
example, Thalassiosira spp. accounted for 24% of the diatom
assemblage at station 30 and Chaetoceros spp. contributed 23%

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3.2.4 Fv/Fm
Distinct changes over time in Fv/Fm were observed between
HL and LL (Figure 6 and Supplementary Table 3). Although Fv/
Fm under LL increased from 0.14 ± 0.02 on day 0 until the end of
incubation, the ratio in HL signiﬁcantly decreased from day 0 to
day 1 (t-test, p< 0.05) and then improved with time. The
maximum values of Fv/Fm reached 0.50 ± 0.03 and 0.61 ± 0.01
on day 7 in HL and the LL, respectively.

3.2.5 P-E parameters
The P-E curve experiments revealed that relationships between
algal carbon ﬁxation rate normalized by chl a concentration andincubation light intensity signiﬁcantly changed during theexperiment (Figures 7, 8 and Supplementary Table 4). The initialslope aB decreased dramatically from day 0 to day 1 in both light
conditions (one-way ANOVA and Tukey-Kramer test, p< 0.05)(Figure 8A), but changed only slightly thereafter. The maximum
carbon ﬁxation rate normalized by chl a concentration (PBmax)
increased until day 4 under HL and LL (Figure 8B). On day 1, PBmax
in the HL condition became lower than in the LL. The
photoacclimation index, Ek, had an increasing trend from day 0
to day 7 and reached 130 and 109 mmol photons m-2 s-1 in HL and
LL, respectively (Figure 8C). The Ek was slightly higher in HL thanin LL throughout the incubation period. The maximum quantum
yield of carbon ﬁxation (Fc max) was relatively high on day 0 (0.018
mol C (mol photon)-1) but sharply declined on day 1 (one-way
ANOVA and Tukey-Kramer test, p< 0.05) and then increased withtime (Figure 8D). Differences in the values of Fc max between HL
and LL were relatively small after day 1, but the value of LL wasslightly higher than that of HL throughout incubation.

FIGURE 2

The composition of viable diatom cells at station 35 by the MPN
method, which was assessed just before the incubation experiment.

half the initial pool size. The carotene pool size (i.e., [carotene]/[chl a]) decreased over incubation (Figure 3F).
The DES value was the highest on day 0 and initially decreasedin HL and LL. After that, there were an increasing trend and adecreasing trend in HL and LL, respectively (Figure 3G).

3.2.3 Diatom cell abundance
The diatom cell abundance increased during incubation(Figure 4 and Supplementary Table 3). The chl a-speciﬁc andthe cell-speciﬁc growth rates were signiﬁcantly different between
the periods from day 0 to day 4 and from day 4 to day 7 in both
HL and LL (one-way ANOVA and Tukey-Kramer test, p< 0.05).
Both of the growth rates were larger in the second half (i.e.,
exponential growth phase) than in the ﬁrst half (i.e., lagphase; Figure 5).
The HL and the LL microscopy data showed that centric
diatoms dominated the microalgal communities, andChaetoceros and Thalassiosira were the main genera (26–79%
and 14–54%, respectively) (Figure 4 and Supplementary
Table 3). On day 0, all Chaetoceros cells were in resting stages(data not shown). While Thalassiosira spp. accounted for a highpercentage of the community from the initial to middle period,
the proportion of C. socialis complex rapidly increased in the
exponential growth phase from day 4 to day 7 under HL and LL.
The other centric diatoms, mainly including D. pumila,
Odontella spp., and P. sulcata, sometimes emerged, but theircontributions were low (1.8–7.6%) in terms of abundance.
Pennate diatoms appeared from all samples, but thecontributions to the diatom communities kept relatively low in
HL and LL (3.6–20% and 5.4–19%, respectively). The differencein the cell concentrations of diatoms between HL and LL on day
7 was not statistically signiﬁcant (t-test, p > 0.05).

Frontiers in Marine Science

4 Discussion
4.1 Critical diatom seed community in
the Chuckchi Sea shelf sediments
The viable diatom cells containing resting stages areaccumulated with high abundance in sediments of thenorthern Bering and the Chukchi Sea shelves (Tsukazaki et al.,2018; Fukai et al., 2019; Fukai et al., 2021). In this study, theconcentrations of viable diatom cells in sediments obtained by
the MPN were similar to those previously reported over the
Paciﬁc Arctic shelves (Fukai et al., 2021). In particular, theregion around station 35, showing the highest concentration of
viable diatom cells in this study, is the so-called biological hot
spots (Grebmeier et al., 2015). Thus the abundant viable diatom
cells may reﬂect the increased productivity in the water column(Fukai et al., 2021). It is also known that lateral advection canenhance particle export in the biological hot spots of the07

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A

B

C

D

E

F

G

FIGURE 3(A–D) The wight-based relative pigment concentration to that on day 0 during the incubation. Fucoxanthin, the sum of chl a and chl c ([chl a] +[chl c]), and total concentrations of measured pigments (chlorophyll a, chlorophyll b, chlorophyll c, chlorophyllide a, 19’butanoyloxyfucoxanthin, fucoxanthin, neoxanthin, diadinoxanthin, diatoxanthin, carotenes) in the high (300 µmol photons m-2 s-1) (A) and low
light (30 µmol photons m-2 s-1) (B) conditions. Light-harvesting pigments (chl a, chl c, plus fucoxanthin; LHP), diadinoxanthin plus diatoxanthin([DD]+[DT]), and carotenes in the high (300 µmol photons m-2 s-1) (C) and low (30 µmol photons m-2 s-1) (D) light conditions. Note that thevalue below 1.0 are showed as the reciprocal value. (E, F) Variations of ([DD] + [DT])/[chl a] (i.e., DD-DT pool size) and [carotenes]/[chl a] (i.e.,
carotene pool size). (G) The de-epoxidation state index (DES; [DT]/([DD]+[DT])) during incubation. Mean values ( ± SD) indicate the average ofthree independent measurements.

C. socialis complex became predominant in the microalgal
communities in terms of abundance with an increase in thewater column stratiﬁcation in the northern Bering and Chukchi
Seas during summer. O’Daly et al. (2020) showed that thesinking POC ﬂux in the Chukchi Sea was among the highest
ever documented across the global oceans, and sinking particles

northern Bering Strait (Cooper et al., 2015). Common diatoms
in the water column of the Paciﬁc Arctic have been observed in
the sediments (Matsuno et al., 2014; Fukai et al., 2020). The
dominant C. socialis complex found at all stations in this study is
known to form dense blooms in the Chukchi Sea from July toAugust (Sergeeva et al., 2010). Suzuki et al. (2021) revealed that

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A

B

FIGURE 4

Cell concentration and composition of diatoms, which were identiﬁed and counted using an inverted light microscope, during incubation under
the high (300 µmol photons m-2 s-1) (A) and low (30 µmol photons m-2 s-1) (B) light conditions. Mean values ( ± SD) indicate the average ofthree independent measurements.

sediments in this study. Tsukazaki et al. (2018) reported thatsome diatoms such as Pseudo-nitzschia delicatissima, Cy.
closterium, Proboscia alata, and Thalassionema spp. dominatedin the autumn water column in the Chukchi Sea cannot survive
in the dark for more than 6 months. Also, few marine pennate
diatoms are known to form resting stages in contrast to centric
diatoms (McQuoid and Hobson, 1996). Therefore, not all
diatoms growing in the water column can survive in the dark
for a long time. However, it is also critical that many “seeds” ofdiatoms exist in the sediments of the Paciﬁc Arctic shelf, andthey can resume even after prolonged dark conditions.

consisting of aggregated diatoms and viable diatom cells wereobserved. Indeed, Chaetoceros spp., including C. socialis
complex, dominated viable diatom communities in sedimentsof the northern Bering and southern Chukchi shelves (Fukai
et al., 2021). Therefore, C. socialis complex, which is capable ofresting stages and settled on the seaﬂoor, can contribute to their
proliferation in the water column once they are brought into
sun-lit surface waters by physical mixing processes. According toLalande et al. (2020), high sinking ﬂux by pennate diatoms of Cy.
closterium and Nitzschia frigida was observed in the Chukchi
Sea. However, the two species were little observed from the

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A

B

FIGURE 5

Cell-speciﬁc (A) and chl a-speciﬁc (B) growth rate during the incubation of two periods (day 0 to day 4 and day 4 to day 7). Light gray and grayrepresent the high (300 µmol photons m-2 s-1) and low (30 µmol photons m-2 s-1) light conditions, respectively. The alphabets indicate
signiﬁcant differences among groups (one-way ANOVA and Tukey-Kramer test, p< 0.05). Mean values ( ± SD) indicate the average of three
independent measurements.

apical axis is 2 mm at the minimum; Hasle and Syvertsen (1997)).
Thus, although there were some uncertainties in cell counting,the enhanced growth of C. socialis complex contributed to theincreased diatom abundance after light exposure.
Light availability is the main trigger for germinating andresuming cell growth from resting stages (Hollibaugh et al.,1981). However, drastic changes in light availability may be
stressful for diatom cells because they need to acclimate to thelight variability. Although b-carotene functions an antioxidant,
the pigment is bound exclusively to photosystems I and II and
not in the peripheral antenna (Wang et al., 2020; Xu et al., 2020;
Buck et al., 2022). Thus, b-carotene can be used as a proxy forthe amount of photosystems (Buck et al., 2022). The cellular
contents of b-carotene in diatoms are reported to be relatively
constant or increase after light exposure (e.g., Kuczynska et al.,2015; Telussa et al., 2019). In the present study, although the
carotene pool size (i.e., [carotenes]/[chl a]) decreased in LL and
HL, the underlying mechanisms are unclear and need to beinvestigated further. To protect the photosynthetic apparatus in
diatoms from high light, the organisms can also utilize thediadinoxanthin-diatoxanthin cycle (Goss and Lepetit, 2015).
Diatom resting stages are reported to have a sufﬁcientxanthophyll pool to support the xanthophyll cycle (Oku and
Kamatani, 1998). In addition, diatoms can alter cellular pigmentcomposition on long time scales of hours to days to acclimate to
the higher light by increasing photoprotective pigments (PPP)
and decreasing light-harvesting pigments (LHP) (Brunet et al.,2011). This study showed temporal changes in DD-DT pool size
between HL and LL. From day 0 to day 1, the DD-DT poolshowed increasing and decreasing trends in LL and HL,
respectively, although both trends were statisticallyinsigniﬁcant. These results suggest that the algal community in
HL suffered from excess light energy for photosynthesis. This
speculation was supported by the results of F v /F m that

4.2 Quick responses for light exposureand high photophysiological plasticity of
viable diatom cells in sediments
The diatom composition on day 0 as counted directly underlight microscopy differed from that estimated with the MPN
method, especially in Thalassiosira spp. and C. socialis complex.
Although the MPN technique can detect viable diatom cells(Imai et al., 1984), the abundance of Thalassiosira spp.
determined by direct cell counting could include their deadcells. In contrast, we could have underestimated the abundanceof C. socialis complex because they have small cell sizes (theFIGURE 6

Variations in the maximum photochemical efﬁciency of PSII (Fv/Fm) during incubation. Mean values ( ± SD) indicate the averageof three independent measurements.

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A

B

C

D

FIGURE 7

Photosynthesis-Energy (P-E) curves during incubation on day 0 (A), day 1 (B), day 4 (C), and day 7 (D) ﬁtted by the photoinhibition model of Platt et al.
(1980). The vertical axis indicates the carbon ﬁxation rate normalized by chl a concentration (mg C [chl a]-1 h-1), and the horizontal one indicates thelight intensity (mmol photons m-2 s-1) during incubation. The red and blue colors represent the high (300 µmol photons m-2 s-1) and low (30 µmol
photons m-2 s-1) light conditions, respectively. Mean values ( ± SD) indicate the average of three independent measurements.

it was the highest on day 0 before light exposure and thendecreased. Diatoms in prolonged darkness can synthesize andaccumulate diatoxanthin (i.e., the so-called dark NPQ), allowing
them to take a low risk of reactive oxygen-dependent damage

dramatically decreased in HL and increased in LL on day 1. It isnoteworthy that even under HL, resting-stages-containing viable
diatom cells can actively resume growing with high Fv/Fm withina few days. Additionally, the DES showed intriguing behavior –

A

B

C

D

FIGURE 8

Variations in photosynthetic parameters during incubation. (A) the initial slope of the P-E curve normalized by chl a concentrations, aB ((mg C[chl a]-1 h-1) (mmol photons m-2 s-1)-1), (B) the maximum photosynthetic rate normalized by chl a concentration, PBmax (mg C [chl a]-1 h-1), (C)the photoacclimation index, Ek (mmol photons m-2 s-1), and (D) the maximum quantum yield of carbon ﬁxation, Fc max (mol C (mol photon)-1).
The red and blue colors represent the high (300 µmol photons m-2 s-1) and low (30 µmol photons m-2 s-1) light conditions, respectively. The
alphabets indicate signiﬁcant differences among groups (one-way ANOVA and Tukey-Kramer test, p< 0.05). Mean values ( ± SD) represent the
average of three independent measurements.

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conditions. The maximum values (4.47 ± 0.740 mg C [mg chl a]h-1 and 4.13 ± 0.213 mg C [mg chl a]-1 h-1 in HL and LL,
respectively) were slightly higher than those from natural Arctic
microalgal assemblages (1.32 ± 0.19 mg C [mg chl a]-1 h-1 and1.29 ± 0.51 mg C [mg chl a]-1 h-1 in 50% and 1% light level,
respectively; Platt et al., 1982) and ice algae (0.052 ± 0.027 mg C
[mg chl a]-1 h-1; Cota, 1985) and the PBmax values of polar
diatoms (0.2–2.46 mg C [mg chl a]-1 h-1; Lacour et al., 2017). The
quick increase in PBmax contrasted with the response of cellabundance and chl a concentration which increased sharply inthe second half of the incubation. Although PBmax generally
increases with increasing temperature (e.g., Yoshida et al., 2018),
the trend may not apply for polar diatoms at −1.6–4.0°C (Lacour
et al., 2017). The PBmax is biophysically determined by the
number of photosynthetic units (n) and the maximum
turnover rate of a photosynthetic unit (t-1) (Sakshaug et al.,1997). Therefore, the dramatic increase in PBmax observed in thisstudy could be attributed to changes in these parameters. Further
study would be required to understand the behavior of PBmax in
Arctic diatoms.

(Lacour et al., 2019; Lepetit et al., 2022). This capability could be
important for diatoms in prolonged darkness because even a low
photon ﬂux might exceed their photosynthetic capacity. The
dark NPQ could provide immediate photoprotection during reillumination (Lacour et al., 2019; Lepetit et al., 2022). In HL, as
the Fv/Fm increased and the DD-DT pool recovered, theincreases in DES were observed, and the diadinoxanthindiatoxanthin cycle might work well, allowing the cells togrow actively.
The quick physiological response to irradiance was also
observed in the carbon ﬁxation, which was similar to theresults of Kvernvik et al. (2018) showing that the highphotosynthetic capacity of microalgae was established within
24 hours of reillumination during the polar night. In this study,
all P-E parameters remarkably changed from day 0 and day 1.
Especially, the drastic decrease in aB and increase in Ek indicatedthat the resting-stages-containing viable diatom cells could
respond to light exposure within 24 hours and acclimate tohigher light conditions for carbon ﬁxation. Since aB can bebiophysically expressed by the functional absorption crosssection of photosystem II (sPSII) and the numbers (n) of
photosynthetic units in photosystem II (i.e., aB=sPSII n )(Sakshaug et al., 1997), the viable diatom cells could decreasesPSII and/or the number of photosynthetic units immediately
after light exposure. The optimum light intensities for carbon
ﬁxation in the vegetative cells under HL and LL during day 4 andday 7 were about 110–130 mmol photons m-2 s-1 as estimated by
Ek. This value could be almost equivalent to those of Arctic
microalgal communities in the euphotic layer (117 and 112 mmol
photons m-2 s-1 in 50% and 1% light level, respectively; using theresults of Platt et al. (1982) with the conversion factor of 4.66mmol photons s-1 per watt used in Cota (1985)). In the Chukchi
shelf, the bottom depths are shallow (ca. 50 m) and euphotic
layers sometimes reaches deeper than 30 m during summer(Suzuki et al., 2021). Therefore, surface sediments, whichcontain abundant diatom resting stages (Fukai et al., 2021),
can easily resuspend into the sunlit layer of the water column bywind events (Uchimiya et al., 2016).
It has been reported that cellular chl a concentrations in
diatom resting stages are relatively high compared withvegetative cells (Hargraves and French, 1983), which would
help diatom resting stages to survive under low light
conditions. In this study, Ek on day 0 was only 4.11 ± 0.28
mmol photons m-2 s-1, indicating their dim-light acclimation.
The Ek value was almost identical to those in Arctic ice algae (2.5
± 1.7 mmol photons m-2 s-1; Cota, 1985). Interestingly, themaximum quantum yield of carbon ﬁxation (Fc max) was
relatively high on day 0 (Figure 8D), suggesting that theresting-stages-containing viable diatom cells in sediments canutilize photons efﬁciently for carbon ﬁxation.
The maximum carbon ﬁxation rate normalized by chl aconcentration (PBmax) became more than double under LL fromday 0 to day 1 and reached the maximum by day 4 in both light

Frontiers in Marine Science1

5 Conclusions
Changes in the sea ice environment in the Paciﬁc Arctic
region can affect light penetration in the water column(Nicolaus et al., 2012; Horvat et al., 2017), resulting inchanges in the composition of microalgae (Ardyna andArrigo, 2020) and viable diatom resting stages in sediments(Fukai et al., 2021). However, few studies have focused on
photophysiological responses of viable diatom in sediments
to light exposure. Previous studies showed the potential
seeding effects of diatom resting stage germination (Shikata
et al., 2009; Hegseth et al., 2019) based on increases in cell andpigment concentrations. In the present study, we revealed
that viable diatom cells containing resting stages wereabundant, and these of Chaetoceros socialis complex were
dominated in sediments of the Chukchi Sea shelf, and they
can resume growing even after the 9 months of darkness.
There was no difference in the cell concentration of diatoms
between LL and HL on day 7, suggesting that the microalgae
were well acclimated to both light conditions. In the higher
latitude region of the Paciﬁc Arctic, there were different viable
diatom communities dominated by Attheya spp., which could
be associated with sea ice (Fukai et al., 2021). Therefore,
further studies are needed to elucidate the photophysiologicalresponses of various diatom communities to light availability.
In this study, the responses of resting-stages-containing
viable diatom cells to light exposure were more quickly inphotosynthetic physiology (i.e., F v /F m , some of the P-E
parameters and DD-DT pool size, and DES) than inabundance (i.e., cell concentration and chl a concentration).
In particular, we found a swift decrease in aB and a gradual12

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increase in Ek (Figure 8), indicating that the resting-stagescontaining viable diatom cells responded to light exposure
within 24 hours and acclimated gradually to higher light
conditions for carbon ﬁxation, respectively. Although theexcess light exposure suppressed the photosynthetic activity
in the microalgal cells at the onset of the experiment, which
was evident by a decreased Fv/Fm of HL on day 0, they
recovered quickly, indicating the high photophysiologicalplasticity to dynamic light changes (Kvernvik et al., 2018).
We also revealed that the optimum light intensities for carbon
ﬁxation in the microalgal cells reactivated from sediments
were about 110–130 mmol photons m-2 s-1, as estimated by Ek
between day 4 and day 7. In the shallow Chukchi shelf, diatom
resting stages in surface sediments may easily resuspend into
the euphotic layer (Uchimiya et al., 2016). The photosynthetic
capability of the viable diatom cells in sediments that quickly
respond to light exposure even after long darkness would
indicate their high seeding potential for blooms in the shallow
Paciﬁc Arctic shelf, which has historically acted as an effective
carbon sink, characterized by tight benthic-pelagic coupling(O’Daly et al., 2020), and is called the “benthic biologicalhotspots” (Grebmeier et al., 2015).

Funding
This work was supported by the Japan Society for the
Promotion of Science (JSPS) KAKENHI Grant Number
JP20J20410 and Arctic Challenge for Sustainability II (ArCS
II) (program grant number JPMXD1420318865) project.

Acknowledgments
We thank the captain, crew, and researchers on board the R/
V Mirai (JAMSTEC) for their tremendous efforts during theﬁeld sampling. We also acknowledge Shigeto Nishino and Koki
Tokuhiro for their support and assistance on the Mirai cruise.
We greatly appreciate the valuable comments provided by Prof.
Douglas Campbell and the two anonymous reviewers.

Conﬂict of interest
The authors declare that the research was conducted in theabsence of any commercial or ﬁnancial relationships that could
be construed as a potential conﬂict of interest.

Data availability statement

Publisher’s note

The original contributions presented in the study areincluded in the article/Supplementary Material. Further
inquiries can be directed to the corresponding authors.

All claims expressed in this article are solely those of theauthors and do not necessarily represent those of their afﬁliatedorganizations, or those of the publisher, the editors and thereviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteedor endorsed by the publisher.

Author contributions
YF conceived this research. YF and AF conducted theﬁeldwork. YF and KS designed the experiment. YF did theexperiment and analyses. YF and KS drafted the manuscript.
YF, KM, AF, and KS contributed to improving the ﬁnal
manuscript. All authors contributed to the article andapproved the submitted version.

Supplementary material
The Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/
fmars.2022.998711/full#supplementary-material

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