RESEARCH PAPER

Spatiotemporal distribution of bacteriochlorophyllsin the meromictic Lake Suigetsu, Japan

Ryuji Kondo • Misa Kodera • Yumi Mori •

Takahiko Okamura • Shinya Yoshikawa •

Kaori Ohki

Received: 15 November 2012 / Accepted: 15 July 2013

� The Japanese Society of Limnology 2013

Abstract The spatiotemporal distribution of chlorophyll

pigments (chloropigments) in the water column of a mer-

omictic lake, Lake Suigetsu (Fukui, Japan), was investi-

gated. Water samples were collected from the central basin

of Lake Suigetsu bimonthly between May 2008 and March

2010 at appropriate depths, including the oxic surface,

oxic–anoxic interface, and anoxic bottom layers. Chloro-

phyll a, related to cyanobacteria and eukaryotic phyto-

plankton, was detected throughout the water column during

the years of the study, whereas bacteriochlorophyll e,

related to brown-colored green sulfur bacteria, was detec-

ted in the anoxic layers below the chemocline at a maxi-

mum concentration of 825 lg L-1. The concentration of

bacteriochlorophyll e was generally maximal at or just

below the chemocline of the lake. The cellular content of

bacteriochlorophyll e was estimated to be low in the upper

part of the chemocline and tended to increase with

increasing water depth. Bacteriochlorophyll a, which was

presumably related to purple sulfur bacteria, was only

detected at the chemocline during summer and autumn at

concentrations of 5.4–16.3 lg L-1. Our analysis of the

chloropigment distribution for the two years of the study

suggested that brown-colored green sulfur bacteria are the

predominant phototroph in the anoxic layers of Lake Sui-

getsu, and that these play a significant role in the carbon

and sulfur cycling of the lake, especially from spring to

summer.

Keywords Bacteriochlorophyll � Chlorophyll �HPLC � Meromictic lake

Introduction

Lake Suigetsu is a typical meromictic lake located on the

coast at Wakasa Bay (which faces the Sea of Japan) in

Fukui, Japan. This lake is part of a chain of five lakes with

a maximum depth of 34 m. Two different types of water

enter the lake—freshwater (through Lake Mikata) and

saltwater (through Lakes Kugushi and Hiruga), so Lake

Suigetsu is a meromictic lake characterized by a permanent

chemocline at a depth of 5–6 m that separates the aerobic

freshwater epilimnion from the anaerobic, saline, sulfido-

genic hypolimnion (Kondo et al. 2000; Matsuyama and

Saijo 1971; Takahashi and Ichimura 1968). Thus, the

deeper layers of the lake stagnate (Matsuyama 1973). With

prolonged meromixis, the anoxic water chemistry of Lake

Suigetsu is dominated by high levels of sulfides (Kondo

et al. 2000; Matsuyama and Saijo 1971).

Lake Suigetsu has attracted interest concerning micro-

bial sulfate reduction and sulfur oxidation in relation to

both the carbon and sulfur cycles because of the charac-

teristics of its water, including the chemocline in the water

column and high concentrations of sulfide in the monim-

olimnion. Thus, we are currently using molecular methods

to investigate the diversity and distribution of sulfate-

reducing bacteria in the water column of Lake Suigetsu

(Kondo et al. 2006; Kondo and Butani 2007). Lake Sui-

getsu is highly sulfidic and the chemocline water is colored

pale red or pink, so it is assumed that this colored water is a

dense bloom of phototrophic sulfur bacteria that is

responsible for microbial sulfur oxidation and carbon fix-

ation. Matsuyama and Saijo (1971) and Takahashi and

Handling Editor: Hideaki Miyashita

R. Kondo (&) � M. Kodera � Y. Mori � T. Okamura �S. Yoshikawa � K. Ohki

Department of Marine Bioscience, Fukui Prefectural University,

Obama, Fukui 917-0003, Japan

e-mail: rykondo@fpu.ac.jp

123

Limnology

DOI 10.1007/s10201-013-0415-y

Ichimura (1968) reported that maximum photosynthetic

bicarbonate fixation took place at the chemocline, and

suggested that sulfur bacteria were involved in the photo-

synthetic carbon assimilation that occurred in that region.

A few studies were carried out to demonstrate the existence

of phototrophic sulfur bacteria in the water column of Lake

Suigetsu, utilizing denaturing gradient gel electrophoresis

(DGGE) of the 16S rRNA gene fragment (Kondo et al.

2009), chlorophyll pigment (chloropigment) analyses

(Okada et al. 2007; Takahashi and Ichimura 1968), and a

functional gene sequence analysis (Mori et al. 2010). More

recently, we demonstrated that brown-colored green sulfur

bacterial counts, as estimated using a new quantitative PCR

method targeting a portion of the a-subunit of the dissim-

ilatory sulfite reductase gene (dsrA), account for up to

35 % of the total bacterial count in the chemocline of Lake

Suigetsu (Mori et al. 2013). However, this molecular

technique for counting populations can detect dead and/or

metabolically inactive microbial cells. On the other hand,

oxygenic pico-sized cyanobacteria were detected at a

maximum density of 4.8 9 105 cell mL-1 even in the

anoxic chemocline of Lake Suigetsu (Okada et al. 2007).

So, it is still unclear which phototrophic microorganisms

are physiologically active in the anoxic layer of Lake

Suigetsu.

Chloropigment analysis potentially provides quantita-

tive estimates of phototrophic bacteria in environments

without the biases attributed to the efficiencies of PCR

amplification and DNA extraction (Nakajima et al. 2003).

Chloropigment analysis has been used for the in situ

characterization of phototrophic sulfur bacterial assem-

blages in stratified water columns (Manske et al. 2005;

Nakajima et al. 2003; Okada et al. 2007). In some cases,

the abundance of phototrophic sulfur bacteria has been

reported as the amount of bacteriochlorophylls (Van

Gemerden and Mas 1995). Such abundances, as reported in

previous studies, vary by several orders of magnitude in the

water column. In the work described in the present paper,

we examined the distribution of phototrophic microbes

using a diagnostic chloropigment analysis to determine the

type of phototrophic population that plays a potential role

in photosynthesis in the anoxic layers of Lake Suigetsu and

to identify spatiotemporal shifts in the phototrophic

populations.

Materials and methods

Samples

Water samples were collected from the central basin of

Lake Suigetsu (35�350 N, 135�530 E) bimonthly between

May 2008 and March 2010 using a Kitahara’s water

sampler (Rigo Co., Ltd., Saitama, Japan) at appropriate

depths, including the oxic surface, oxic–anoxic interface,

and anoxic bottom layers. The samples were immediately

poured into brown bottles to prevent light exposure. All

samples were kept in an ice-cooled box and transported to

the laboratory within a few hours of sampling. Tempera-

ture, salinity, and dissolved oxygen (DO) concentration

were measured using oxygen meters (Model 85; YSI,

Yellow Springs, OH, USA and HQ30d; HACH, Loveland,

CO, USA). The vertical turbidity profile (as kaolin

mg L-1; ppm) was obtained using a turbidity meter (model

PT-1; JFE Advantec Co., Ltd., Kobe, Japan). Vertical

profiles of photosynthetically active radiation (PAR,

400–700 nm) were measured using an LI-192SA under-

water quantum sensor and an LI-1400 datalogger (LI-COR,

Lincoln, NE, USA).

Chemicals and chloropigment preparation

All chemicals used in this study were of analytical grade or

HPLC grade. Chlorophyll a (Chl a) and bacteriochloro-

phyll a (BChl a) were purchased from Wako Pure Chem-

ical Industries (Osaka, Japan) and Sigma–Aldrich Fine

Chemicals (St. Louis, MO, USA), respectively. The bac-

teriochlorophyll e (BChl e) used in this study was extracted

from Chlorobium phaeovibrioides DSM 269T, which was

obtained from Deutsche Sammlung von Mikroorganismen

und Zellkulturen GmbH (DSMZ) and cultured in our

laboratory using DSMZ medium 29 (http://www.dsmz.de/

microorganisms/medium/pdf/DSMZ_Medium29.pdf). These

chloropigments were dissolved in diethyl ether and

used for calibration. The chloropigment concentrations

in the standard solutions were calculated using molar

extinction coefficients of 8.98 9 104 M-1 cm-1 for Chl

a at 661 nm, 9.70 9 104 M-1 cm-1 for BChl a at 771 nm,

and 3.4 9 104 M-1 cm-1 for BChl e at 647 nm (Takamiya

et al. 2003), after performing absorption spectrum mea-

surements using a PharmaSpec UV-1700 spectrophotom-

eter (Shimadzu, Kyoto, Japan).

Pigment extraction

A 100-mL water aliquot was filtered through a 25-mm GF-

75 glass fiber filter (nominal pore size, 0.3 lm; Advantec,

Tokyo, Japan) to collect suspended particulate matter for

subsequent chloropigment extraction. The filters were

stored in a 2-mL screw-cap plastic tube at -135 �C in the

dark until processed. The filter samples were suspended in

1 mL of acetone and sonicated (10 W) for 1 min on ice.

The samples were then centrifuged at 20,0009g for 15 min

at 4 �C and the supernatant was filtered through a 0.45-lm

Cosmonice Filter S (Nacalai Tesque, Kyoto, Japan). A

portion (500 lL) of the filtrate was evaporated in vacuo

Limnology

123

using a centrifugal vacuum evaporator (Iwaki VEC-100;

AGC Techno Glass, Chiba, Japan). After evaporation, the

chloropigment was redissolved in 50 ll of acetone. All

processes were performed under dim light conditions.

High-performance liquid chromatography

and quantification

The acetone extract was analyzed via high-performance

liquid chromatography (HPLC), as described previously

(Okada et al. 2007) with some modification. Briefly, the

HPLC system consisted of a Class LC-VP/SPD-M10AVP

system (Shimadzu, Kyoto, Japan). Chloropigments were

separated with a Wakosil-II 5C18HG column

(250 9 4.6 mm i.d.; Wako Pure Chemical Industries,

Osaka, Japan) and a guard column, and detected with a

photodiode array detector (SPD-M10AVP; Shimadzu). All

analyses were performed at 40 �C with a flow rate of

1 mL min-1 for 60 min. Methanol was used as the solvent.

Aliquots of 20-ll chloropigment extracts or standard

solutions were injected into the HPLC system. Chromato-

grams were digitally recorded on a computer using the

CLASS-VP chromatography data system (Shimadzu). The

chloropigment concentrations were quantified at wave-

lengths of 431 nm for Chl a, 770 nm for BChl a, and

473 nm for BChl e. Three BChl e homologs can be

detected by our HPLC procedure (Okada et al. 2007).

Concentrations of BChl e were calculated by summing the

three HPLC peaks.

Results

Water column profiles

Figure 1 shows seasonal changes in the depth distributions

of the physicochemical properties in the central basin of

Lake Suigetsu during the sample collection times. The

epilimnion temperature in Lake Suigetsu ranged from 3 �C

in the winter to 30 �C in the summer. The hypolimnion

temperature was, however, constant at ca. 14 �C through-

out the year. The salinity was 2–4 practical salinity units

(psu) for the epilimnion water and 12–15 psu for the

hypolimnion water. Surface water was saturated with DO,

and the DO concentration decreased rapidly below 3–5 m

to below the detection limit at 5–8 m and down to the

bottom at 34 m. This observation shows the stagnation of

the anoxic saline water in deeper layers from approxi-

mately 6 m to the bottom at 34 m. Turbidity was 2–8 ppm

in the surface layer and increased to its maximum at the

oxic–anoxic interface zone. Turbidity decreased with depth

between 9 and 25 m, remained constant from 25 m to

30 m, and below that increased slightly with depth.

Distribution of chloropigments in the water column

Chlorophyll a (Chl a) was detected in all samples,

including those from the oxic surface, oxic–anoxic inter-

face, and anoxic bottom layers, at concentrations ranging

from 3.5 to 80.6 lg L-1 (Fig. 2). High concentrations of

Chl a were observed in the surface water in January and

March in both years. Apart from the high concentrations

observed in the epilimnion in winter, Chl a concentrations

ranged from 3.6 to 16.8 lg L-1 in the surface water and

increased to maxima of 5.6–11.6 lg L-1 at the chemo-

cline, and then gradually decreased with water depth

between the vernal and autumnal seasons (Fig. 2).

Bacteriochlorophyll a (BChl a) was detected in the

redox boundary waters between July and November at low

concentrations ranging from 5.4 to 16.3 lg L-1 (Fig. 2).

Bacteriochlorophyll e (BChl e) was detected in and

below the chemocline during all sampling occasions at

high concentrations ranging from 21.6 to 825 lg L-1,

whereas BChl e was not detected in the oxic surface layer

(Fig. 2). The concentrations of BChl e changed with

depth and/or season. A high concentration of BChl

e ([100 lg L-1) was generally detected in metalimnetic

water during the warm season (from July to November),

whereas the concentration decreased from November to

March during the next year.

Discussion

Chlorophyll a

In the surface water of Lake Suigetsu, according to our

microscopic observations, dinoflagellate blooms of Het-

erocapsa occur during the winter season, while cyano-

bacteria such as Microcystis and Anabaena and/or diatoms

dominate during the summer season. High concentrations

of Chl a in the winter season appear to correspond with the

Heterocapsa bloom. The vertical profiles of Chl a concen-

tration with a broad peak at the chemocline correspond to

those of pico-sized cyanobacterioplankton within the Syn-

echococcus/Cyanobium clade (Okada et al. 2007; Ohki

et al. 2012). Ohki et al. (2012) isolated sulfide-tolerating

pico-sized cyanobacteria from the chemocline of Lake

Suigetsu. However, considering the low concentration of

Chl a, the cyanobacteria seem to be less abundant and to

have lower photosynthetic activities than anoxygenic

phototrophs in the chemocline of Lake Suigetsu.

Bacteriochlorophyll a

Purple sulfur bacteria have been reported to form dense

blooms in the chemoclines of meromictic lakes similar to

Limnology

123

Lake Suigetsu (Bosshard et al. 2000a, b; Tonolla et al.

1999). However, our chloropigment analysis suggested that

purple sulfur bacteria comprise only a minor fraction of

photosynthetic prokaryotes in the chemocline of Lake

Suigetsu. Purple sulfur bacteria generally require high light

intensity (Van Gemerden and Mas 1995). In Lake Suigetsu,

the chemocline usually developed at a depth of around

4–7 m (Fig. 1; Kondo and Butani 2007; Kondo et al. 2000,

2006, 2009; Okamura et al. 2012). PAR at the chemocline

was as low as 0.01–16.2 lmol m-2 s-1 at water depths of

between 5 and 7 m, which is insufficient for the growth of

purple sulfur bacteria of genus Chromatium (Van Gemer-

den and Mas 1995). Therefore, relatively high light inten-

sities during the summer season may cause the occasional

appearance of purple sulfur bacteria in the chemocline of

the lake.

BChl a is a chloropigment that is known to occur in

small amounts in green sulfur bacterial cells (Imhoff 1995).

If the BChl a detected in the chemocline is from green

sulfur bacteria, the ratio of the concentration of BChl a to

that of BChl e should be constant within the water samples

analyzed. However, BChl a was detected only in the

chemocline during warmer seasons, and was not always

detected in the water samples in which BChl e was

detected. Furthermore, the relationship between the con-

centration of BChl a and that of BChl e was examined by

Spearman’s rank and Peason’s correlation analyses, but no

correlation was found. Relatively large cells (more than

5 lm in length) with sulfur-globule-like inclusions in the

cells were observed microscopically during our investiga-

tion, especially during warmer seasons. Purple sulfur bac-

terial genes associated with the family Chramtiaceae were

occasionally detected in the chemocline of Lake Suigetsu

during clone library analysis of the PCR products of the

dsrA gene (Mori et al. 2013). In addition to these findings,

as BChl a is the major photosynthetic pigment of purple

sulfur bacteria, it seems that purple sulfur bacteria are the

main source of the BChl a detected in the chemocline of

Lake Suigetsu, rather than green sulfur bacteria.

Bacteriochlorophyll e

Green sulfur bacteria have been reported to be able to grow

under low light intensity by changing their pigment com-

position (Manske et al. 2005). Moreover, it has been shown

that brown-colored green sulfur bacteria have a relatively

high BChl e content as an adaption to low-light conditions

(Overmann et al. 1992; Repeta and Simpson 1991). These

Dep

th o

f wat

er (

m)

Water temperature (°C)

A

27.5

2522.52017.5

1515

12.5107.5

17.5

20

22.5 25

12.5107.5

5

Salinity (psu)

B

4 46

68 10

12

14

Dissolved oxygen (mg L-1)

C

14 1012468

141612

102

0

Turbidity (ppm)

D

2

3

4

5

10 20 30 4050

2

3

45

2

3

45

10203010

30

223

3 3 3 3

3

4

0

5

10

15

20

25

30

0

5

10

15

20

25

30

0

5

10

15

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25

30

0

5

10

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25

30

010290028002M J J A S O N D J F M A M J J A S O N D J F M

010290028002M J J A S O N D J F M A M J J A S O N D J F M

010290028002M J J A S O N D J F M A M J J A S O N D J F M

010290028002M J J A S O N D J F M A M J J A S O N D J F M

Fig. 1 Seasonal changes in the vertical distributions of a water temperature, b salinity, c dissolved oxygen, and d turbidity in Lake Suigetsu

between May 2008 and March 2010. Dots indicate measurements on specific dates and at particular depths

Limnology

123

observations indicate that chloropigment concentrations in

natural environments do not necessarily represent the cell

density of phototrophic sulfur bacteria. The relationship

between the BChl e concentration and the phototrophic

sulfur bacterial density as determined by a quantitative

real-time PCR (qPCR) targeting the dsrA gene (Mori et al.

2013) was examined using Spearman and Pearson corre-

lation analyses, but only a weak correlation was found

(q = 0.516, p = 0.061 for the Spearman test and

r = 0.576, p = 0.031 for the Pearson test). This weak

correlation suggests that the BChl e detected in the

chemocline of Lake Suigetsu originates from green sulfur

bacteria.

The high concentrations of BChl e are within the range

of, or higher than, values previously reported for other

meromictic lakes (Borrego et al. 1997; Nakajima et al.

2003; Van Gemerden and Mas 1995 and references

therein). Green sulfur bacteria of Chlorobium, Prosthec-

ochloris, and Pelodictyon have been detected mainly in or

just below the chemocline of Lake Suigetsu by DGGE

analysis of 16S rRNA gene fragments (Kondo et al. 2009)

and clone library analysis of PCR products of the dsrA

gene (Mori et al. 2010; 2013). These results, together with

our chloropigment determination, indicate that brown-col-

ored green sulfur bacteria form the dense bacterial layer in

the chemocline of Lake Suigetsu. In general, the dominant

species of green sulfur bacteria is Chlorobium phaeobac-

teroides in meromictic freshwater lakes or Chlorobium

phaeovibrioides and Pelodictyon phaeum in meromictic

brackish waters (Borrego et al. 1997). Phototrophic sulfur

bacterial assemblages in the saline meromictic Lake Sui-

gesu coincide with this general trend in species habitat

segregation.

Two groups of phototrophic sulfur bacteria, purple sul-

fur bacteria and green sulfur bacteria, inhabit similar

environments, where anoxic layers containing reduced

sulfur compounds are exposed to light. However, due to

differences in the physiological characteristics (such as

optimum temperature, light illumination/wavelength, and

electron donor concentrations for growth) between the two

groups (Van Gemerden and Mas 1995), environmental

conditions may determine the dominant group of

1

5

6

7

10

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6

7

10

1

5

6

7

10

1

5

6

7

10

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6

7

10

100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103

100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103 100 101 102 103

1

5

6

7

10

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10

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6

7

10

Dep

th o

f wat

er (

m)

Chloropigment concentration (µg L-1)

Chloropigment concentration (µg L-1)

May 2008 July 2008 September 2008 November 2008 January 2009 March 2009

May 2009 July 2009 September 2009 November 2009 January 2010 March 2010

Fig. 2 Seasonal changes in the vertical distributions of chlorophyll

a (open bar), bacteriochlorophyll a (stippled bar), and bacteriochlo-

rophyll e (cross-hatched bar) in the water column of Lake Suigetsu

between May 2008 and March 2010. Error bars represent the

standard error of the mean (n = 3)

Limnology

123

phototrophic sulfur bacteria. The dominant population of

phototrophic sulfur bacteria varies depending on the lake

considered (Borrego et al. 1997; Bosshard et al. 2000a, b;

Koizumi et al. 2004; Nakajima et al. 2003; Øvreas et al.

1997; Tonolla et al. 1999). In meromictic Lake Kaiike,

similar to Lake Suigetsu, purple sulfur bacteria formed a

narrow band at the upper part of the chemocline, while

green sulfur bacteria were found underneath (Nakajima

et al. 2003). This spatial segregation of purple and green

sulfur bacteria was not observed in Lake Suigetsu. Among

the phototrophic sulfur bacteria, the brown-colored green

sulfur bacterial species were able to adapt, grow, and

dominate in the chemocline of the lake.

Cellular content of bacteriochlorophyll e

The cellular content of BChl e in green sulfur bacterial

cells may vary depending on the depth and season of the

sample. Using the same samples as those employed for our

chloropigment analysis, we roughly determined the cellular

content of BChl e using the BChl e concentration deter-

mined in this study and the cell density of phototrophic

sulfur bacteria as gauged by qPCR (Mori et al. 2013). As

shown in Table 1, the cellular content of BChl e was

estimated to be low in the upper parts of the chemocline

and tended to increase with increasing water depth. Trace

amounts of BChl e were detected in the samples from 6 m

depth in May 2008 and 6 m in January 2009, where high

densities of phototrophic sulfur bacteria were detected, but

these amounts of BChl e were below the limit of detection

(ca. 1 lg L-1). Conversely, the dsrA gene of phototrophic

sulfur bacteria was below the limit of detection (103

cells mL-1) in the sample taken at a depth of 10 m in

September 2008, where BChl e was detected at a concen-

tration of 67 lg L-1. In general, the green sulfur bacteria

appear more able to adapt their light-harvesting systems by

increasing their specific contents of pigments (Van

Gemerden and Mas 1995). It has been shown that brown-

colored green sulfur bacteria have a relatively high BChl

e content as an adaptation to low-light conditions (Over-

mann et al. 1992; Repeta and Simpson 1991). Light is of

crucial importance for the growth and activity of photo-

trophic sulfur bacteria in stratified lakes (Guerrero et al.

1985). Changes in the cell-specific content of BChl e with

water depth can be explained by differences in PAR irra-

diance at the depth at which green sulfur bacteria live.

Green sulfur bacteria in deeper layers of Lake Suigetsu

may maintain high levels of photosynthetic pigments to

allow them to grow under low-light environments. Fur-

thermore, the cellular content of bacteriochlorophylls var-

ies with species (Van Gemerden and Mas 1995). Our clone

library analysis of the dsrA gene showed minor changes in

green sulfur bacterial assemblages with water depth and

season (Mori et al. 2013). Changes in the composition of

the green sulfur bacterial population also influence the

estimated cellular content of BChl e. Thus, changes in the

cell-specific content of BChl e may be influenced by both

the physiological state and the composition of the green

sulfur bacteria.

Conclusion

Using chloropigment analysis, we have shown the vertical

and temporal shifts in the concentrations of photosynthetic

pigments along the water column of Lake Suigetsu over a

two-year sampling period. The dominant chloropigment

was BChl e in and below the chemocline, indicating that

brown-colored green sulfur bacteria were the principal

component of the photosynthetic microorganisms present

in the anoxic waters of the lake. Water at the chemocline of

Lake Suigetsu is colored pale red or pink, especially during

the summer season. Matsuyama and Saijo (1971) reported

that the maximum photosynthetic carbon assimilation took

place at the chemocline, and they suggested that purple

sulfur bacteria were responsible for photosynthetic carbon

Table 1 Calculated bacteriochlorophyll e (BChl e) contents in green

sulfur bacterial cells, along with BChl e concentrations and green

sulfur bacteria (GSB) cell densities

Date of

sampling

Depth

(m)

BChl

e conc

(lg L-1)

GSB densitya

(cells mL-1)

BChl

e content

(pg cell-1)

21 May 2008 6 ND 1.3 9 104 NA

7 187 1.6 9 106 0.11

10 73 8.3 9 104 0.88

12 July 5 ND 1.4 9 104 NA

6 554 3.1 9 106 0.18

7 746 7.4 9 105 1.00

10 52 8.5 9 103 6.21

18 September 6 22 7.7 9 105 0.03

7 445 2.3 9 105 1.98

10 67 ND NA

6 November 5 26 3.9 9 104 0.65

6 825 1.5 9 106 0.54

7 290 3.1 9 104 9.34

10 35 1.2 9 104 2.88

6 January 2009 6 ND 2.2 9 104 NA

7 69 7.8 9 104 0.88

10 44 7.4 9 104 0.59

4 March 10 51 4.3 9 104 1.19

ND not detected, NA not availablea Cell counts of green sulfur bacteria were estimated assuming that

all cells had only a single copy of the dsrA gene, and were obtained

through qPCR quantification

Limnology

123

assimilation in this zone. However, brown-colored green

sulfur bacteria species are responsible for the pale-red

colored water of the chemocline; they appear to be active

and play an important role in carbon assimilation and

sulfide oxidation in Lake Suigesu. Although chloropigment

analysis is useful for the in situ characterization of photo-

trophic sulfur bacterial assemblages in nature, it remains to

be determined whether the green sulfur bacteria contribute

to carbon assimilation in Lake Suigetsu as well as the

oxidation of reduced sulfur compounds. Molecular analy-

ses such as stable-isotope probing and mRNA quantifica-

tion of functional genes could be useful in this respect.

Acknowledgments We are grateful to N. Kawahara and J. Butani

from our laboratory for their assistance with the field sampling. We

also gratefully acknowledge the constructive comments of anony-

mous reviewers and the handling editor and their suggestions for

improving this paper. This study was supported in part by a Fukui

Prefectural Fund for the Promotion of Science to R.K.

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