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Short Communication
Characterization of a Marine Bacterium Passing through a 0.1-μm Pore-
sized Filter
Haruo Yamaguchi1*, and Kazumasa Yamada2
1Faculty of Agriculture and Marine Science, Kochi University, Monobe-Otsu, Nankoku, Kochi 783–8502, Japan; and 2Faculty of
Marine Science and Technology, Fukui Prefectural University, Gakuen-cho, Obama, Fukui 917–0003, Japan
(Received February 20, 2024—Accepted November 14, 2024—Published online March 8, 2025)
OPEN ACCESS
Citation: Yamaguchi, H., and Yamada, K. (2025)
Characterization of a Marine Bacterium Passing through a
0.1-μm Pore-sized Filter. Microbes Environ 40: ME24014.
https://doi.org/10.1264/jsme2.ME24014
*Corresponding author. E-mail: yharuo@kochi-u.ac.jp
Copyright: © Japanese Society of Microbial Ecology /
Japanese Society of Soil Microbiology / Taiwan
Society of Microbial Ecology / Japanese
Society of Plant Microbe Interactions /
Japanese Society for Extremophiles
https://creativecommons.org/licenses/by/4.0/
The present study aimed to isolate and characterize a marine bacterium
capable of passing through a 0.1-μm pore-sized filter (0.1-μm filter).
Sediment suspension samples were filtered through 0.1-μm filters, inoculated
into sterile media, and incubated. Isolated SspURN76 belonged to
Saccharospirillum, according to 16S rRNA gene sequencing, and showed a
very slender shape. The minimum cell size of SspURN76 was 0.09×3.2 μm.
These morphological features of SspURN76 were likely responsible for its
passage through 0.1-μm filters. Based on the results obtained herein, marine
bacteria may be present in 0.1-μm filtered fractions.
Key words: bacteria, filters, Saccharospirillum, slender, 0.1 μm
Marine bacteria play central roles in global biological and
chemical cycles. Filters with pore sizes ≤0.45 μm have been
used to collect and/or remove bacterial cells in seawater
through filtration procedures (MacDonell and Hood, 1982;
Hoff, 1993; Kirchman, 2012). However, not all bacterial
cells are trapped by these filters (Duda et al., 2012; Nakai et
al., 2013; Obayashi and Suzuki, 2019).
Bacteria have a number of morphological features that
contribute to their passage through filters with micropores.
MacDonell and Hood (1982) reported that some bacterial
populations passed through filters with a pore size of
0.2 μm. Nakai et al. (2013) filtered coastal waters though
0.2-μm filters and then isolated many species of
Saccharospirillum, Reinekea, and other genera from the fil‐
trates. These small and filterable bacteria show great diver‐
sity, as summarized by Ghuneim et al. (2018) and Nakai
(2020). However, to the best of our knowledge, there is no
evidence of marine bacterial isolates passing through not
only 0.2-μm filters, but also 0.1-μm pore-sized filters (0.1-
μm filters).
In a freshwater ecosystem, Wang et al. (2007) found that
approximately 0.2% of freshwater bacterial cells were capa‐
ble of passing through 0.1-μm filters (Fig. S1). Hylemonella
gracilis (Betaproteobacteria) was isolated from 0.1-μm fil‐
trates (Wang et al., 2008). The cells of this isolate had the
smallest width (0.2 μm), a slender shape, and passed
through a 0.1-μm filter (Wang et al., 2008).
In consideration of these issues and marine bacterial
diversity, we aimed to confirm the hypothesis that some
marine bacterial populations pass through 0.1-μm filters.
The present study isolated and characterized a marine bacte‐
rium that passes through not only 0.2-μm filters, but also
0.1-μm filters and discussed its morphological features.
Sampling was conducted on the coast of Tosa Bay in
Kochi Prefecture, Japan. On July 13, 2020, sediments were
collected from the station (33°26′09.6″N, 133°27′36.0″E)
using an Ekman–Birge-type bottom sampler (RIGO). The
surface sediment (0–2 cm) was sampled using a dispensing
spoon and transferred into a plastic container. The sample
was stored in the dark at –25°C prior to isolation.
Some of the sediment sample was suspended in auto‐
claved seawater and centrifuged for 1 min using a manual
centrifuge 1011 (Hettich) with a swing-out rotor 1025
(Hettich). The resultant supernatant was used for bacterial
isolation. Cultures of Karenia papilionacea (Dinophyceae)
KpNOM1H (Yamaguchi et al., 2016b) were also used. Pre‐
liminary to the present study, we found bacterial growth in a
0.2-μm filtered sample of the culture. These samples were
respectively filtered through a 0.2-μm filter (Minisart NML;
Sartorius) and then through a 0.1-μm filter (Milex-VV;
Merck Millipore). Syringe top-type filters were used in the
filtration process. The membrane materials of the 0.2-μm
and 0.1-μm filters were cellulose acetate and polyvinylidene
fluoride (PVDF), respectively.
Filtrate samples originally obtained from coastal
sediments were inoculated into axenic clonal cultures of the
diatom, Chaetoceros sp. (unidentified). Cultures were main‐
tained in SWM-3 (Chen et al., 1969) with 2 nM Se (Imai et
al., 1996). Culturing was conducted at 20°C with 100–
120 μmol photons m–2 s–1 (cool white fluorescence) on a
12:12 h light:dark cycle. Cell suspensions were filtered as
described above.
Serially diluted (10–1–10–11) samples of the 0.2-μm and
0.1-μm filtrates were prepared with autoclaved seawater.
Samples were then inoculated into test liquid media, which
contained 0.5 g L–1 tryptone (Nacalai Tesque) and 0.05 g L–1
Microbes Environ. 40(1), 2025
https://www.jstage.jst.go.jp/browse/jsme2 doi:10.1264/jsme2.ME24014
Article ME24014
dried yeast extract (Nacalai Tesque). After an incubation at
20°C in the dark, growing bacterial samples were spread
onto black-stained test agar media containing 2% (w/v)
black color powder (Kyoritsu foods). Solid cultures were
then incubated in the dark at 20°C. According to the streak
plating method, a bacterial colony was picked, inoculated
into the test liquid medium, and then established as the pure
culture isolate.
The present experiments investigated the sequence of the
16S rRNA gene region. Cells of the strains were harvested
by centrifugation at 8,200×g for 10 min. Genomic DNA
extraction, PCR, and sequencing procedures were per‐
formed by Macrogen Japan Corp. Genomic DNA was
extracted using PrepMan Ultra Sample Preparation Reagent
(Thermo Fisher Scientific). The target region of DNA was
amplified using PrimeSTAR HS DNA polymerase
(TaKaRa) with 50 μM of oligonucleotide primer sets: 27F
(5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-
TAC GGY TAC CTT GTT ACG ACT T-3′). PCR mixtures
were prepared according to the manufacturer’s instructions.
According to the procedures of Macrogen Japan Corp., the
targeted fragments were amplified with thermal cyclers.
Amplified PCR fragments were verified using agarose gel
electrophoresis against known standards and then purified
using the ExoSAP-IT Express PCR Product Cleanup Reagent
(Thermo Fisher Scientific). Sequencing reactions were per‐
formed in a Bio-Rad C1000 or S1000 thermal cycler using
the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo
Fisher Scientific), according to the manufacturer’s instruc‐
tions. Single-pass sequencing was performed on each tem‐
plate using 518F (5′-CCA GCA GCC GCG GTA ATA
CG-3′) or 800R (5′-TAC CAG GGT ATC TAA TCC-3′) pri‐
mers. Fluorescent-labeled fragments were purified from the
unincorporated terminators either by ethanol precipitation or
using the BigDye XTerminator Purification Kit (Thermo
Fisher Scientific). Samples were analyzed using a 3730xl
DNA Analyzer (Thermo Fisher Scientific).
The resulting sequences were assembled using MEGAX
v10.1.7 (Tamura et al., 2011). A single consensus sequence
(1,473 bp) of the strain was elucidated. The 5′ and 3′
ends were manually aligned to truncate and refine both
ends. The sequences of Saccharospirillum species and
closely related species used in this study were obtained from
the NCBI database (Fig. 1). Sequences were aligned using
the CLUSTAL_W algorithm (Thompson et al., 1997). Open
and extended gap penalties were set at 10.0 and 5.0, respec‐
tively, for both the pair-wise and multiple alignment phases.
Divergent rates in the completed alignments among
Saccharospirillum species and strains were estimated using
simple uncorrected pair-wise distance (p distance) matrices
in MEGAX v10.1.7 (Tamura et al., 2011).
Maximum-likelihood (ML) analyses were conducted with
1,000 bootstrap replications using MEGAX ver. 10.1.7. The
best-fit model for ML was obtained with partial (95%)
deletion; no significant differences were noted in the best-fit
model or phylogeny between the partial deletion and com‐
plete deletion approaches. In ML analyses, the Kimura 2-
parameter and gamma distribution (G+I) model were used
for 16S rRNA gene regions.
Posterior probabilities for the Bayesian inference (BI)
were calculated using MrBayes 3.1.2 (Huelsenbeck and
Ronquist, 2001; Ronquist and Huelsenbeck, 2003) and the
posterior probability distribution was estimated using the
Metropolis-Coupled Markov Chain Monte Carlo (MCMCMC)
method. MCMCMC from a random starting tree was used in
this analysis with two independent runs, one cold chain, and
three heated chains with the temperature set at 0.2. Trees
were sampled every hundredth generation for more than one
million generations. To increase the probability of chain
convergence, more than 500 trees were sampled after the
Strain SspURN76 [LC795725]
Saccharospirillum correiae [KY310592]
Strain NOW [AB540009]
Strain kure [AB540008]
Strain SmNOM1 [LC834167]
Saccharospirillum mangrovi [MF850374]
Saccharospirillum aestuarii [GQ250189]
Saccharospirillum salsuginis [EF177670]
Saccharospirillum impatiens [AJ315983]
Saccharospirillum alexandrii [MH197114]
Reinekea marina [KC690143]
0.01
-/100
-/75
-/99
1.00/99
1.00/100
1.00/96
-/100 -/98
1.00/56
1.00/55
1.00/79
Salinisprillum marinum [KJ195687]
Reinekea marinisedimentorum [AJ561121]
Reinekea aestuarii [GQ456131]
Reinekea blandensis [DQ403810]
Saccharospirillum Reinekea
Fig. 1. Maximum likelihood phylogenetic analysis of Saccharospirillum and other genera based on 16S rRNA gene sequences. Bayesian
posterior probability values are shown with bootstrap percentage values (n=1,000) from a maximum-likelihood analysis at each branching
position; a hyphen indicates that branching was not supported in Bayesian analyses. The strains isolated in the present study and their accession
numbers are shown in bold font. Reineke and Salinispirillum species are used as the outgroup genera close to Saccharospirillum.
Yamaguchi and Yamada
2 / 6 Article ME24014
standard deviations of the two runs decreased to <0.01 to
calculate posterior probability. The number of burn-ins was
500.
Bacterial cells were fixed with glutaraldehyde (1% [v/v],
final concentration) for a morphological analysis. Fixed
cells were collected on a 0.05-μm filter (111103 Whatman
nuclepore track-etched membranes; Cytiva). The resultant
filter was air-dried and mounted on scanning electron
microscopy (SEM) specimen stubs, which were coated with
osmium (thickness of 10 nm) using an osmium coater
(Osmium Plasma Coater OPC60A; Filgen). Cell images
were obtained using SEM (SU1510; Hitachi).
In the whole-mount analysis, 5 μL of the fixed cell sus‐
pension was dropped onto a formvar-coated copper grid.
After 5 min, excess liquid was removed with filter paper,
and a drop of 15 to 30× diluted EM stainer (Nisshin EM)
was placed on the grid for 5 s and then removed. After air
drying, the specimen was observed using transmission elec‐
tron microscopy (TEM) (HT7700; Hitachi).
The width and length of individual bacterial cells were
calculated based on measurements from TEM images.
According to equation (1) shown in Wang et al. (2008), the
cell volume of bacterial cells was calculated as follows:
Cellvolume =4
3πr3+πr2L−2r , (1)
where r represents half of the smallest width and L repre‐
sents the length of the bacterial cell.
The effect of the filtration volume on bacterial passage
through a 0.1-μm filter was examined. Before and after fil‐
tration, the numbers of bacterial living cells were measured
using the most probable number (MPN) method. Two bacte‐
rial strains, Phaeobacter sp. URN3 (Yamaguchi et al.,
2016a) and Saccharospirillum sp. SmNOM1 (present
study), were used as controls. The former had no ability to
pass through 0.2-μm or 0.1-μm filters, whereas the latter
isolated from the K. papilionacea culture passed through
0.2-μm filters, but not 0.1-μm filters. As these strains prolif‐
erated in liquid media, the cultures became white and
cloudy. Cloudiness before and after filtration was assessed.
Bacterial cultures that grew well in liquid media were
diluted (10×) using fresh media. After an incubation for 1 h,
10, 20, and 40 mL of the cell suspensions were filtered
through 0.1-μm filters. In the cases of URN3 and
SmNOM1, 20 mL of the cell suspensions were used. The
0.1-μm filters were the track-etched, nucleopore, and poly‐
carbonate membrane types (Whatman 111105; Cytiva).
With a hand pump (MV8510; Mityvac), filtration was con‐
ducted under a pressure of less than 10 inHg (254 mmHg).
Filtrates were serially diluted at 10–1–10–11 as described
above, and then cultured in quintuplicate in 48-well clear
plates (Iwaki) under dark conditions at 20°C for 5 d. Unfil‐
tered cell suspensions (whole culture) were used as the con‐
trol. The number of positive wells in which bacterial growth
appeared was counted. Combinations of positive and nega‐
tive wells were used to construct a statistical table. These
procedures were used to calculate MPN.
In the present study, the marine bacterial strain,
SspURN76, which is capable of passing through 0.1-μm fil‐
ters, was isolated. Prior to inoculation into the sterile
medium, the sediment suspension (original sample) was fil‐
tered through 0.1-μm filters. Liquid cultures showed a
slightly white and cloudy color, and bacterial colonies
appeared on the black-stained agar plate. In contrast,
bacterial cultures of Phaeobacter sp. URN3 and
Saccharospirillum sp. SmNOM1 did not proliferate under
the same filtration procedures. Notably, SmNOM1 cultures
grew well when filtered through 0.2-μm filters.
SspURN76, along with SmNOM1, belonged to the genus
Saccharospirillum (Gammaproteobacteria) in the molecular
phylogenetic tree (Fig. 1). Partial 16S rRNA gene sequences
of SspURN76 were matched by >98% with those of
Saccharospirillum correiae and Saccharospirillum mangrovi;
the p distance was closer to the former (0.00987) than to the
latter (0.01802) among Saccharospirillum strains (Table
S1). SspURN76 was genetically different (p distance:
0.01652), with SmNOM1 being incapable of passing
through the 0.1-μm filter (Table S1). Additionally, the p dis‐
tance was close (0.00450) between Saccharospirillum
alexandrii and Saccharospirillum impatiens.
TEM images displayed slender cells that possessed a long
flagellum (Fig. 2). Cell width and length were 0.09–0.19
and 2.16–4.70 μm, respectively (n=30, Figs. 2 and 3). The
minimum cell size of SspURN76 was 0.09×3.2 μm (Fig. 3).
Saccharospirillum species, except for S. salsuginis, are
spirillum-shaped bacteria. Among Saccharospirillum spe‐
cies, SspURN76 cells showed a similar filamentous slender
shape to that of S. mangrovi cells (Fig. 3). A comparison of
minimum cell widths between S. mangrovi (0.3 μm) and
SspURN76 (0.09 μm) confirmed that SspURN76 cells were
slender. Assuming the slender and spirillum-like cell shape,
the cell volume of SspURN76 was estimated to be 0.022–
0.095 μm3 (n=30). Saccharospirillum sp. SspURN76 ap‐
peared to be an ultra-slender marine bacterium with a cell
volume <0.1 μm3.
We herein describe the marine bacterial strain,
Saccharospirillum sp. SspURN76, which is capable of
passing through a 0.1-μm filter. The cells of both
Saccharospirillum sp. SspURN76 (present study) and H.
gracilis (Wang et al., 2008) showed a slender filamentous
shape. As reported by Nakai (2020), Silvanigrella paludirubra
and Fluviispira multicolorata recently described by Pitt et
al. (2020) are in a category of slender filamentous bacteria
and appear to be rod- and/or filamentous-shaped bacteria
with widths >0.2 μm (see Fig. 1 in Pitt et al., 2020). To the
best of our knowledge, it has not yet been established
whether S. paludirubra and F. multicolorata are capable of
passing through 0.1-μm filters. We consider a bacterial slen‐
der shape similar to a hair to be decisive in the passage of
bacteria through filters. Wang et al. (2008) showed that H.
gracilis cells had not only the smallest width of 0.2 μm, but
also a slender shape. Furthermore, SspURN76 cells were
slender with a minimum width of 0.09 μm. These findings
suggest that the ultra-slender shape of SspURN76 is a criti‐
cal factor for its passage through a 0.1-μm filter.
The MPN of the tested SspURN76 cultures was
0.98×108 mL–1, which is close to the density of living cells
(Fig. 4). Most, but not all, living cells passed through
0.1-μm filters depending on the filtration volume, and 28%
of all cells passed through these filter when 40 mL of cul‐
ture was filtered (Fig. 4). The filtration volume affected the
Marine Bacterium through 0.1-μm Pores
3 / 6 Article ME24014
passage of Saccharospirillum sp. SspURN76 through the
0.1-μm nuclepore filter (the present study), which is consis‐
tent with previous findings on H. gracilis (Wang et al.,
2008). This phenomenon may be attributed to the change in
the bacterial concentration directly above the filter surface
(Wang et al., 2008). Cell concentrations above the filter sur‐
face increase when retaining bacterial cells and increasing
the filtration volume; bacterial cells may then be pushed
and/or push other cells into micropores.
Bacterial cell sizes and their passage through filters with
1.2
Cell width (μm)
S. impatiens
S. salsuginis S. correiae
S. aestuarii
S. mangrovi
10.80.60.40.20
12
10
8
6
4
2
0
14
Cell length (μm)
S. alexandrii
Fig. 3. Cell width and length of Saccharospirillum species (shaded
square areas), including SspURN76 (white square area). Circles
indicate cell length and the smallest width of individual cells of
SspURN76 (n=30). Size data are referred to as follows: S. aestuarii
(Choi et al., 2011), S. alexander (Yang et al., 2020), S. correiae
(Fidalgo et al., 2017), S. impatiens (Labrenz et al., 2003), S. mangrovi
(Zhang et al., 2018), and S. salsuginis (Chen et al., 2009).
micropores vary. Some field populations in coastal waters
initially pass through 0.2-μm filters, but subsequently lose
this passage in nutrient-enriched media due to cell enlarge‐
ment (Obayashi and Suzuki, 2019). Torrella and Morita
(1981) showed an increase in the size of freshly incubated
bacterial cells. This study used culture media that allowed
10
8
10
7
10
6
10
9
Whole
10 20 40
Filtration volume (mL)
Bacterial cell density (MPNs mL
-1
)
Fig. 4. Living-cell densities of Saccharospirillum sp. SspURN76
cultures diluted (10×) using fresh media, incubated for 1 h, and
filtrated through a 0.1-μm pore-sized filter. A serial dilution method
(quintuplicate) calculated living cells as the most probable number
(MPN). Error bars show 95% confidence intervals.
Fig. 2. Cell images of Saccharospirillum sp. SspURN76 cultures shown by scanning electron micrographs (A and B) and whole-mount
transmission electron micrographs (C, D, E, and F).
Yamaguchi and Yamada
4 / 6 Article ME24014
Saccharospirillum sp. SspURN76 and other species (Urata
et al., 2022) to proliferate well. SspURN76 cells in the
media clearly exhibited a nutrient-repleted state and were
able to pass through 0.1-μm filters (Fig. 4). The present
results strongly suggest that the passage of SspURN76
through a 0.1-μm filter is not transient, but may vary among
individual cells.
Bacterial passage through micropores of filters may be
affected by cell states and filtration procedures. Cell shape,
size, flexibility, and elasticity as well as the vacuum pres‐
sure and sample volume at the filtration procedure are
factors that may affect bacterial passage. Therefore, compa‐
ratively examining the morphological and physiological
states of SspURN76 cells in various filtration procedures
will be necessary in the future to understand bacterial pas‐
sage through micropore filters.
To remove bacterial cells and prepare extremely small
particles, such as diatom viruses (size 0.02–0.04 μm), from
aquatic environments, our laboratory occasionally freezes
sediment suspensions and/or water samples, filtrates the
thawed samples through 0.2-μm and 0.1-μm filters, and then
inoculates the filtrates into axenic diatom cultures. In our
experience, cultures rarely become cloudy during incuba‐
tions. To obtain bacterial cells from such a fraction, we ino‐
culated the sediment sample into algal cultures and isolated
Saccharospirillum sp. SspURN76. No algicidal activity of
the bacterium culture was detected during these procedures.
The freezing of samples and culturing them with diatom
cells may affect the isolation of SspURN76.
Marine bacteria, such as Saccharospirillum sp.
SspURN76, may contribute to filtration sterilization failure.
Cells of Saccharospirillum species have widths >0.3 μm
(Fig. 3) and, thus, are unlikely to pass through filters with
micropores. Nakai et al. (2013) isolated Saccharospirillum
kure and NOW (Fig. 1 and Table S1) from 0.2-μm filtered
seawaters collected at Hiroshima and Okinawa, respectively.
Furthermore, the present study showed that SmNOM1
passing through a 0.2-μm filter was closely related to
Saccharospirillum kure (Fig. 1). Therefore, some
Saccharospirillum species appear to pass through filters
with 0.2-μm and/or 0.1-μm pores and may contribute to
filtration sterilization failure. However, these failures are
infrequent. Previous studies, particularly those that
employed sterilization with various chemicals and seawater
samples, reported that 0.2-μm and/or 0.1-μm filtration pro‐
cedures were effective. Specifically, filtration procedures
using 0.1-μm filters ensured sterilization in most cases.
Marine bacterial cells trapped on 0.2-μm filters, and even
on 0.1-μm filters, are unlikely to represent the full spectrum
of microorganisms present. To obtain a comprehensive
understanding of microbes in marine environments, future
studies that focus on monitoring marine bacteria in not only
0.2-μm, but also 0.1-μm filtrates are needed. The present
results indicate the potential of Saccharospirillum sp.
SspURN76 as a standard for 0.1-μm pore filterable bacteria.
Acknowledgements
This study was supported by JSPS KAKENHI (Grant Number:
JP22K19210).
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