Rikunomizu（Limnology in Tokai Region of Japan）87 : 1 - 9（2020）

ArticleWhat caused the pink coloration of water at the bottom of Lake Shiraishi ?
Masaki KIHIRA1), Suguru OKUNISHI2), Tadashi INAGAKI3),
Susumu KATO1) and Hiroto MAEDA 2)

Abstract
Pink water was collected from the bottom of Lake Shiraishi, a brackish lake located in Kihoku-cho, Mie
Prefecture, in September 2009 and January 2010 for preliminary field investigation. The water quality
and bacterial community structure were investigated again in August 2010 to clarify the cause of the pinkwater; microscopic observation to clarify the morphology and PCR-denaturing gradient gel electrophoresis(DGGE) to characterize bacterial community were performed. The water sampled from near the bottomof the lake at a depth of 8 m was colored pink and smelled of hydrogen sulfide. Microscopic examination
revealed that the dominant cells were 5–7 µm in size, spherical or ovoid, pink to purple in color, with gas
vesicles, and were not motile. The results of microscopy and phylogenetic analysis using PCR-DGGE
suggested that the pink coloration was related to photosynthetic sulfur bacteria of either Halochromatium
roseum or Lamprobacter modestohalophilus.
Key words: Photosynthetic sulfur bacteria, PCR-DGGE, Lake Shiraishi(Received: 30 May 2020; Accepted: 18 July 2020)

Microbial communities at layers where the dissolved
oxygen gradient changes are interesting from the viewpointof degradation of organic compounds in the redox system,
and analysis of these communities using 16S rRNA genes
is advancing. Kondo et al. (2009) reported that green
sulfur bacteria phylogenetically related to the genera
Prosthecochloris, Pelodyctyon, and Chlorobium within
the phylum Chlorobi, were dominant in the chemocline inLake Suigetsu; furthermore, Lunina et al. (2007) reported
that green sulfur bacteria, purple sulfur bacteria, and purple
nonsulfur bacteria were detected in Lake Shunet, Khakassia.
In Lake Shiraishi, several studies have been conducted
for continuous measurement of water temperature, salinity,
dissolved oxygen (Tsuchihashi et al. 1996), and bacterial
community in the sediment (Santander-De Leon et al.

Introduction
Lake Shiraishi, located in Kihoku-cho, Mie, Japan, isa brackish lake covering an area of 472,000 m2 with amaximum depth of 9 m (Tsuchihashi et al., 1996). The lake
receives freshwater input from the Funatsu and Choshi
rivers, and seawater input from Hikimoto Bay. Brackishlakes have a complex water environment with a mixture of
fresh and seawater. In Japan, lakes Suigetsu (Kondo et al.,2009; Mori et al., 2013), Kaiike (Matsuyama 2004), Obuchi(Ueda et al., 2000), Abashiri (Mikami et al., 2002), andothers have been reported in studies on brackish lakes. The
characteristics of these brackish lakes throughout the year
are that density gradients appear and the bottom of the lake
is consistently extremely hypoxic.
1)

Iga Research Institute, Mie University, 3-3, 1 cho-me, Yumegaoka, Iga, Mie, 518-0131, Japan
Research Field in Fisheries, Agriculture Fisheries and Veterinary Medicine Area, Research and Education Assembly, Kagoshima University, 4-50-20
Shimoarata, Kagoshima, 890-0056, Japan
3)
Kousui Co., Ltd., 17-2, Hiedanocho Rokuya Kamo, Kameoka, Kyoto, 621-0037, Japan
Corresponding author: Suguru OKUNISHI, E-mail: okunishi@fish.kagoshima-u.ac.jp
2)

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Masaki KIHIRA et al.

2013a; 2013b).
Oyster farming is a flourishing industry in this lake and is
the main fishing industry present. It was reported that large
numbers of oysters died during the summer (Tsuchihashi
et al. 1996). So, we conducted some basic research into thereason for these deaths in our laboratory (Santander-De Leonet al. 2013a). During this investigation, we encountereda pink-colored water. Even those that have been involvedin the aquaculture industry for decades do not know about
this coloring phenomenon, which suggests that it is notoften observed. The purpose of this study was to clarify thecause of the pink water from an ecological viewpoint. To
achieve this aim, microorganisms were characterized by the
denaturing gradient gel electrophoresis (DGGE) method andmicroscopic examination.

measured using an automatic water analyzer (HYDROLAB
DS5, Kankyo System). Water column samples werecollected along a vertical distribution every 1 m fromthe surface to the bottom (water depth 8 m: 9 layers)
to measure nutrient contents and bacterial community
composition at the sampling station using a motor pump.
Water samples collected for chemical analysis of nutrientswere immediately filtered through glass fiber filters (GF75, Advantec) that had been precombusted at 420℃ . Water
samples collected for dissolved silica were immediately
filtered through quantitative filter paper (No. 5C, Advantec).
Filtrates for determination of nutrients and dissolved silica
were stored at -20℃ and 4℃ , respectively, until chemical
analysis in the laboratory. Nutrients were measured using an
auto analyzer (SWAAT AASU-2000, BLTEC). Dissolved
silica concentrations were analyzed spectrophotometricallyusing the molybdenum blue method of Saijo and Mitamura(1995). Bacterial cells were observed under a microscope
without treatment (BZ-9000, Keyence). For PCR-DGGE
analysis, the bacterial cells were collected by filtration. We
selected a pore size of 3.0 µm to focus on pink-coloredbacteria with a large cell size (>3.0 µm) and investigate theirdistribution. 30 mL water samples (depth of 0 m to 7 m) and10 mL (depth of 8m) were filtered through a polycarbonate
membrane filter (3.0 µm, Advantec) to collect bacterial

Methods
Sampling station and sample treatments

The sampling station was located at the center of Lake
Shiraishi, where the oyster farming was concentrated(Fig. 1). A preliminary field investigation was conductedin September 2009 and January 2010, and the main fieldsampling commenced in August 2010. Water temperature,salinity, dissolved oxygen and chlorophyll a (Chl. a) wereN

Funatsu RiverstationLake Shiraishi

Choushi River

Hikimoto Bay
㻞㻜㻜㻌㼙Fig. 1. The location of the sampling station at Lake Shiraishi, Mie, Japan (34 6'47.6"N; 136 14'16.4"E).

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2What caused the pink coloration of water at the bottom of Lake Shiraishi ?

cells. The membrane filter was stored at -20 ℃ until DNA
extraction. DNA extraction was carried out according to the
manufacturers’ instructions (QuickGene DNA tissue kit S,
KURABO) using the QG-Mini80 extraction machine. The
DNA concentration was measured based on the absorbancevalue at 260 nm using absorption spectrometer (DU730,
BECKMAN COULTER).

200 µL TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM
ethylenediaminetetraacetic acid), and frozen and thawed toelute the amplified DNA. The excised single bands were reamplified using the same PCR conditions with the reaction
volume of 20 µL, and the amplicons were electrophoresedon 1.5% agarose gel at 100 V. The PCR products were
purified in the same method as for DGGE analysis. The
sequences were determined with ABI PRISM 3500xl
Genetic Analyzer (Applied Biosystems, Carlsbad, CA,USA), and the BigDye Terminator Cycle Sequencing Kit
Ver. 3.1 (Applied Biosystems) was used in accordance withthe manufacturer’s instructions. The sequencing primer was
the 341F primer without a GC clamp.

PCR amplification of the 16S rRNA gene

PCR amplification of 16S ribosomal RNA genes was
carried out using the forward primer 341F (5’CCTACGGGAGGCAGCAG-3’) with a GC-clamp and thereverse primer 907R (5’- CCGTCAATTCCTTTGAGTTT-3’)(Muyzer et al. 1995) in 25 µL reaction volumes, whichcontained 0.6 units of Ex Taq (Takara Bio, Otsu, Japan), 2.5µL of dNTP mixture (2.5 mM each), 2.0 µL of each primer (5pmol each), 11.0 µL of DNA-free water, and 5 µL of DNA
template (containing 1–10 ng of DNA). The thermal cycling
conditions were optimized using a touchdown program as
follows (Don et al., 1991): after initial denaturation of 5 min
at 95℃ , 19 cycles of 95℃ for 1 min, 62℃ for 1 min, and
72℃ for 1 min, decreasing the denaturation temperature after
the first cycle by 0.8 ℃ every cycle followed by nine cyclesof 95 ℃ for 1 min, 52 ℃ for 1 min, and 72 ℃ for 1 min; afinal cycle of 95℃ for 1 min, 55℃ for 1 min, and 72℃ for
10 min for final extension was conducted. The amplicons
were confirmed using agarose gel electrophoresis. PCR
products were purified using the Wizard SV Gel and PCR
Clean-Up System (Promega, Madison, WI). Purified DNA
concentrations were measured in the same method as for
DNA extracts.

Phylogenetic analysis

Approximately 500 bp of each DGGE band sequence
was used for the phylogenetic analysis. The phylogenetic
positions of the excised bands were determined by searching
the DDBJ database with the BLAST program (http://blast.
ddbj.nig.ac.jp/blastn?lang=ja), and the sequences werealigned using the CLUSTAL X program (version 2.1)
(Larkin et al., 2007). A phylogenetic tree was constructed by
the neighbor-joining method with 1,000 bootstrap replicatesin CLUSTAL X (Saitou and Nei, 1987).
Accession numbers

The partial nucleotide sequences of the 16S rRNA genefragments are available in the DDBJ database underaccession numbers LC543551 to LC543564.

Results
When the water was collected at a depth of 8.5 m, the water
was pink in color and smelled of hydrogen sulfide during
the preliminary investigation in September 2009. The water
samples collected at each depth in September 2009 areshown in Fig. 2. The pink color was evident from a depth of
7.5 m and was the darkest at 8.5 m in September 2009 (Fig.
2-A); even in winter (January 2010) the water was pink at
7.5 m depth (Fig. 2-B). In August 2010, the water takenfrom 8 m was pink and smelled of hydrogen sulfide. Verticalprofiles of the water temperature, salinity, dissolved oxygen,
and Chl. a were shown in Fig. 3. The water temperature in
the 1.5 m layer was slightly higher than that in the surface
layer and was almost constant below 1.5 m. A steep salinity
gradient was observed between 0 and 1 m, with little change
from about 1.5 m down. The vertical profile of dissolved

DGGE conditions

DGGE was performed by using a D-Code system (BioRad Laboratories) according to Muyzer et al. (1993, 1995).
Approximately 700 ng of the purified PCR products wereapplied to 6% polyacrylamide gel with urea and deionized
formamide with a gradient concentration range from 25%
to 50% (where 100% denaturing gels contain 7M urea and
40% formamide in 0.5× TAE buffer). Electrophoresis was
run at 60V, 60 ℃ for 16 h. Gels were stained with SYBRGold (Thermo Fisher, USA) and observed on a blue lighttransilluminator.
Sequence analysis of DGGE bands

DGGE bands were excised from the gels using Pasteur
pipettes. The excised gel pieces were suspended in

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Masaki KIHIRA et al.

A

8.5 m

8m

B

7.5 m

7m

6m

4m

2m

1m

0m

Fig. 2. Photographs of the water collected during the preliminary
investigation. A: vertical sampling of water from 0 to 8.5 m(September 2009), B: comparison of water at depths of 7.5 m (left)
and 7 m (right) (January 2010).

oxygen was almost uniform from 0 m to 6 m, and decreased
sharply deeper than approximately 6.5 m. The average
concentration of Chl. a from the surface to 5 m depth was
about 1.7 µg L-1, while the value increased with depth andthe maximum was about 7.9 µg L-1 in the 8 m layer.
The nutrient concentrations in the water samples areshown in Table 1. The concentrations of ammonia andphosphate were extremely high at the lake bottom. The
nitrite concentration was distributed almost uniformly fromthe surface to the lake bottom. The nitrate concentration in
the surface water was markedly high and was distributed
uniformly in layers deeper than 1 m. The silica concentrationin the surface water was high and was extremely high at thelake bottom.
Salinity (psu)

Dissolved oxygen (mg L-1)

Chl.a (mg L-1)

Depth (m)Water temperature （℃）

Fig. 2
15
Fig. 3. Vertical distribution of water temperature, salinity, dissolved oxygen and the concentration of chlorophyll a (Chl. a)in August 2010.

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4What caused the pink coloration of water at the bottom of Lake Shiraishi ?

Microscopic observation showed that the dominant cells
in the pink colored lake bottom water were coccoid or
ovoid, approximately 5–7 µm in length with some vesicles
in the enteric cytoplasm. Cells were either purple or pink incolor (Fig. 4). No flagellum was found, and no motility wasobserved.
The DGGE banding patterns of the microbial populations
at different depths are shown in Fig. 5. The band pattern
was very similar among samples from 0–6 m in depth. The
pattern below 7 m was different when compared with theupper layers; in particular, the band pattern of 8 m depth was
greatly different from that of the other layers. Four bands(bands 2, 3, 4 and 6) showed dominance in the upper layerfrom 6 m. Bands that were unique at the depth of 8 m wereobserved from the DGGE profile. DNA sequences wereobtained from a total of 14 bands on the DGGE gels. The
closest strain and phylogenetic group of each sequence arelisted in Table 2. Bands 1–3 and 5 were derived from thechloroplast of phytoplankton. Bands 4 and 6 that dominatedfrom 0 to 7 m depth were uncultured bacteria belonging
to Bacterioidetes and Ralstonia insidiosa, respectively, ofthe β-proteobacteria. Bands 8–11, which appeared only
at 8 m depth, were closely homologous to the green sulfur
bacterium, Chlorobium spp. Band 14 was very similar
to both Halochromatium roseum and Lamprobacter
modestohalophilus belonging to the purple sulfur bacteria,with the same similarity to each strain in the BLAST search.
The phylogenetic tree containing sequencing results of the
DGGE bands and the closest strains is shown in Fig. 6.
The microorganisms detected by DGGE were chloroplast
derived from phytoplankton, Chlorobi, Bacteroidetes,
Chloroflexi, β-proteobacteria, and γ-proteobacteria. Band
14 showed the same homology value (99.4%) for both L.
modestohalophilus and H. roseum, but the phylogenetic
tree indicated that band 14 was more closely related to L.
modestohalophilus.

Table 1. Nutrient concentrations in water samples at eachsampling depth in August 2010 (N.D. = not detected).
Depth
（m）

NH4+ - N(mg L-1)

NO2- - N(mg L-1)

NO3- - N(mg L-1)

PO43- - P
(mg L-1)

Si(mg L-1)

0

0.013

0.001

0.026

N.D.

2.16

1

0.012

0.001

0.006

0.002

0.409

2

0.010

N.D.

0.005

0.003

0.481

3

0.018

0.001

0.006

0.004

0.407

4

0.026

N.D.

0.010

0.003

0.270

5

0.015

N.D.

0.005

0.027

0.186

6

0.035

0.002

0.011

0.041

0.315

7

0.051

0.001

0.004

0.126

0.383

8

1.46

0.001

0.005

0.579

4.14

Fig. 4. Observation of microorganisms in samples taken frompink-colored water (8 m layer) of Lake Shiraishi in August2010 using a microscope.

1m 2m 3m 4m 5m

0m

1
2

3

6m

7m

8m

5
8

4

6

9
10
11

7

Fig. 417

12
13

Discussion

14

Fig. 5. Denaturing gradient gel electrophoresis profiles ofuniversal 16S rRNA gene amplicons of each layer of Lake
Shiraishi. Open circles with numbers indicate excised bands.
These bands were analyzed by sequencing, with the results ofsequencing shown in Table 2.

Fig. 5

One of the major characteristics of brackish water lakes isthat seawater and freshwater are intricately mixed, which
greatly affects the distribution of chemical water quality andthe biology of microorganisms (Clark, 1977; Barnes, 1980).
The water temperature and salinity profiles indicated thata halocline was formed as a result of freshwater flowing
from the river during the investigation (Fig. 3). The highconcentrations of nitrate and silica in the surface water

18

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Masaki KIHIRA et al.
Escherichia coli strain K-12 (NC_010473)
793
999
1000

Band 1 (LC543551)

0.02

Chaetoceros calcitrans, chloroplast (FJ002215)
chloroplast

Band 2 (LC543552)
Band 3 (LC543553)
Chaetoceros simplex, chloroplast (KJ958479)

Band 5 (LC543555)
1000 Pararotalia nipponica, chloroplast (KY498712)
Band 4 (LC543554)

1000

693

1000
961

Uncultured Flavobacteriales (LC314465)
Band 7 (LC543557)
Bacteroidetes

831 Uncultured Bacteroidetes (AB266010)
Haliscomenobacter hydrossis (NR_042316)

1000

Band 12 (LC543562)

815

Uncultured bacterium (KX177108)

1000
1000
732

Maribellus luteus (NR_165017)
Band 8 (LC543558)
Prosthecochloris vibrioformis (AM690794)
Band 9 (LC543559)

1000 Chlorobium phaeobacteroides (CP001101) Chlorobi
Chlorobium limicola (KX417793)
Chlorobium luteolum (CP000096)

1000

Band 11 (LC543561)

988

Band 10 (LC543560)
Band 13 (LC543563)
1000 Uncultured Chloroflexi (FM242343)

859

Chloroflexus aurantiacus (NR_043411)
1000

Chloroflexi

Chloroflexus aggregans (MN173283)

Band 6 (LC543556)
1000 Ralstonia insidiosa (LT548965)

β-Proteobacteria

Chromatium minus (Y12372)
Thiocapsa pendens (AJ002797)
Band 14 (LC543564)
979

Lamprobacter modestohalophilus (HQ877095)
999
889

γ-Proteobacteria

Halochromatium roseum (NR_042529)
Halochromatium salexigens (X98597)
Halochromatium glycolicum (X93472)

Fig. 6

Fig. 6. Neighbor-joining tree of partial 16S rRNA gene sequences recovered from denaturing gradient gel electrophoresis
bands. Accession number appears after the genus name. The scale 19bar indicates 0.05 changes per nucleotide. The numberson the branches refer to bootstrap values for 1,000 replicates; only those above 650 are shown. Escherichia coli was used
as an outgroup.
Table 2. Basic local alignment search tool (BLAST) homology searches of denaturing gradient gel electrophoresis bands
obtained from Lake Shiraishi.
DGGE band

Sequence Identity(%)

Taxonomic group

Accession no.

1

chloroplast

505/507

(

99.6%

)

chloroplast

FJ0022152

chloroplast

494/495

(

99.8%

)

chloroplast

JN207230

3

chloroplast

493/506

(

97.4%

)

chloroplast

KJ958479

4

Uncultured bacterium

517/517

(

100%

)

Bacteroidetes

JQ198577

5

chloroplast

503/505

(

99.6%

)

chloroplast

KY498712

6

Ralstonia insidiosa517/517

(

100%

)

β-Proteobacteria

LT548965

7Uncultured bacterium

510/510

(

100%

)

Bacteroidetes

GQ916124

8

Prosthecochloris vibrioformis

513/513

(

100%

)

Chlorobi

AM690794

9Chlorobium phaeobacteroides

508/508

(

100%

)

Chlorobi

CP001101

10

Chlorobium limicola

484/504

(

96.0%

)

Chlorobi

KX417793

11

Chlorobium luteolum

493/504

(

97.8%

)

Chlorobi

CP000096

12

Uncultured bacterium

500/512

(

97.7%

)

Bacteroidetes

KX177108

13

Uncultured Chloroflexi bacterium

488/495

(

98.6%

)

Chloroflexi

FM242343

Halochromatium roseum

521/524

(

99.4%

)

Lamprobacter modestohalophilus

521/524

(

99.4%

)

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Most homologus relatives

The Tokai Branch of the Japanese Society of Limnology

6γ-Proteobacteria

NR042529
HQ877095

What caused the pink coloration of water at the bottom of Lake Shiraishi ?

also supported this fact. Conversely, the concentrations ofammonia and phosphate were high at the lake bottom (Table1). The tendency of these vertical distributions was similar
in various brackish water lakes, such as Lake Nakaumi(Kondo et al., 1994) and Lake Abashiri (Mikami et al.,2002). In addition, the dissolved oxygen was consumed in
the bottom layer and the oxygen concentration was greatly
reduced near the bottom of the lake.
Similar to the distribution of photosynthetic bacteria
in the chemocline in Lake Suigetsu (Mori et al., 2013)
and in the upper part of the anoxic layer in Lake Abashiri(Mikami et al., 2002), the gradient in oxygen concentration
affected the microbial community composition in Lake
Shiraishi. The microbial community in the 8 m layer where
dissolved oxygen was low was very different from thatof the other layers, which was consistent with the otherfeatures of water from this layer (pink colored and smelledof hydrogen sulfide). Pimenov et al. (2003) reported a pale
pink layer of water because of the development of purple
bacteria (6 × 105 cells mL-1), which were assigned by theirmorphological and spectral characteristics to L. purpurea
in Lake Shira (Khakassia). In the present study, the numberof cells that caused the pink color shown in Figure 4 could
not be accurately counted but the total bacterial count by
DAPI (4’,6-diamidino-2-phnylindole) staining was on theorder of 106 cells mL-1, and many of the cells were larger
than 3 µm (data not shown). Considering the water quality
environment at a depth of 8 m, a high percentage of thebacteria in the lake bottom water are perhaps photosynthetic
bacteria. Therefore, photosynthetic bacteria are the most
likely candidates responsible for the pink color. In fact, theChl. a concentration was elevated near the bottom layer,
but this might have been because of photosynthetic bacteria
capable of producing bacteriochlorophyll a rather than byphytoplankton.

Although a halocline was formed near the surface andthe salinity changed significantly, the banding pattern wasalmost the same from the surface to a depth of 7 m except forband 5 (Fig. 5). In other words, the flow of water from theriver had no significant effect on the microbial community
composition collected by the 3 µm pore size filter. In
contrast, the community composition was drastically alteredin the lake bottom water with low oxygen. This implies
that the gradient of dissolved oxygen concentration had asignificant effect on the microbial community composition.
The results of the DGGE sequencing showed the presenceof bacteria of purple sulfur bacteria of the γ-Proteobacteria
and green sulfur bacteria of the phylum Chlorobi, in the 8 m
layer (Fig. 5, Table 2), indicating that the gradient of oxygen
concentration greatly affected the microbial community
composition. Normally, the cells of Chlorobi group, whichhave a small cell size, would pass through the 3µm filter.
However, the filter was clogged with pink bacteria andsuspended solid when the cells were collected. It is possiblethat the cells of Chlorobi were trapped with other bacteria.
In the 8 m layer, the intensities of bands 8 and 14 werestronger than those of the other bands. Erroneous results canoccur when PCR-DGGE analysis is used to quantify the
microbial community in the environment (Nakagawa andFukui, 2002). However, it has also been reported that whena high percentage of cells of a particular microorganism arepresent in a microbial community, the strength of the DGGE
band corresponding to that microorganism was also strong(Casamayor et al., 2000). In addition, Okunishi et al. (2012)
showed that the abundance of microorganisms was reflectedin the strength of the band. Therefore, the abundance of
Prosthecochloris vibrioformis (band 8), a green sulfur
bacterium, and L. modestohalophilus or H. roseum (band14), a purple sulfur bacterium, are likely to be higher thanthose of other microorganisms inhabiting the lake bottom

Table 3. Comparison of the characteristics of Halochromatium roseum, Lamprobacter modestohalophilus andthe cells of this study.“+”and“−”stand for the presence or absence of a feature. Data for reference species
were taken from Kumar et al. (2007) (H. roseum strain JA134T) and Gorlenko and Imhoff (2005) (L.
modestohalophilus).
Characteristic

H. roseum

L. modestohalophilus

This study

Cell shape

Rod

Ovoid to rod

Spheriacl or Ovoid

Motility－+－

Cell size (µm)

2.0–3.0×3.0-5.0

2.0 – 2.5

5–7

Gas vesicles

+

+

+

Colour of cell suspension

Purple-pink

Purple-red

Purple-pink

NaCl optimum (%)

1.5 – 2.5

1–4

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Masaki KIHIRA et al.

water, based on the density of bands 8 and 14. Among them,
the pink color of the lake bottom water suggested that thebacteria that appeared as band 14, which is closely related topurple sulfur bacteria, made a significant contribution.
Sequence analysis of band 14 showed that it was closely
related to two bacteria, L. modestohalophilus and H. roseum,with the same homology (Table 2). However, the resultsof the phylogenetic tree showed that band 14 was moreclosely related to L. modestohalophilus (Fig. 6). Since theresults of microscopic observation reflected the results of
the nucleotide sequence and phylogenetic tree, band 14 wasconsidered to be originated from the cells observed in Fig.
4. Table 3 summarizes the morphological characteristics
of L. modestohalophilus (Gorlenko and Imhoff, 2005), H.
roseum (Kumar et al., 2007) and the characteristics of thebacteria collected from Lake Shiraishi as determined frommicroscopic observations. The cells in Lake Shiraishi wereoval-shaped with a diameter of 5–7 µm and were larger
than those of both H. roseum and L. modestohalophilus.
Additionally, the cells were growing at salinity of about
30 psu, and motility was not observed. Consequently, it
was morphologically difficult to identify them as either H.
roseum or L. modestohalophilus.
In this study, it was revealed that the pink water takenfrom the bottom layer of Lake Shiraishi in August 2010
was caused by photosynthetic sulfur bacteria, H. roseum
or L. modestohalophilus. However, neither H. roseum
nor L. modestohalophilus could be confirmed geneticallyor morphologically. To confirm the identity of thisphotosynthetic bacterium, it is necessary to isolate andcharacterize this photosynthetic bacterium from the field andexamine it in more detail using primers specific to sulfur
photosynthetic bacteria.

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Acknowledgments
We thank Mr. Hiroya AZECHI for providing informationon oyster aquaculture and taking samples at Lake Shiraishi.
This work was undertaken thanks to the research grants of
Chubu Electric Power Co., Ltd. and Daiei THA Co., Ltd.
We express our gratitude to both companies.

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(Editor: Professor Dr. Jun MURASE, Nagoya University)摘要

白石湖におけるピンク色の湖底水の要因は何か？
紀平征希1)・奥西将之2)・稲垣直史3)・加藤

進1)・前田広人2)

三重県紀北町にある汽水湖の白石湖において2009年9月および2010年1月の予備調査で，湖底付近からピンク色の水が採水された。このピンク色の水を解明するために，2010年8月に再度調査を行い，同時に顕微鏡観察，PCR-DGGE 解析を行った。その結果，水深8m の湖底付近から採水した水はピンク色で硫化水素のにおいがした。また顕微鏡観察では，細胞大きさは5-7 µm で球形または卵形をしており，色はピ
ンクから紫色，ガス胞を持ち，運動性はなかった。顕微鏡観察の結果と PCR-DGGE の解析結果から，ピンク色の要因はHalochromatium roseum または Lamprobacter modestohalophilusのどちらかの光合成硫黄細菌のよるものだと考えられた。
キーワード：光合成硫黄細菌，PCR-DGGE，白石湖
1)〒518-0131三重県伊賀市ゆめが丘1-3-3三重大学伊賀研究拠点

2)〒890-0056鹿児島県鹿児島市下荒田4-50-20鹿児島大学学術
研究院農水産獣医学域水産学系

3)〒621-0037京都府亀岡市稗田野町鹿谷加茂17-2 株式会社こうすい
連絡著者：奥西将之（E-mail: okunishi@fish.kagoshima-u.ac.jp）

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Ⓒ2020

The Tokai Branch of the Japanese Society of Limnology