Cultivation of microbial communities from a hot spring and interaction among microbes involved in sulfur cycling

修 士 学 位 論 文

題 名

Cultiv劉tionofmicrobialcommHnitiesfyomahotspring andinter負ctionamongmicrobesinvolvedinsulfurcycling

邦題:温 泉微生物群集の実験室内での培養 と硫黄循環に関与する微生物の相互関係(英 文)

指導教授 松浦 克美 教授

平 成23年1月7日 提 出

首都大学東京大学院

理工学研究科 生命科学専攻 学修番号09881333

氏 名 原 なつみ

温 泉 微 生 物 群 集 の 実 験 室 内 で の 培 養 と 硫 黄 循 環 に 関 与 す る 微 生 物 の 相 互 関 係

環 境微 生物 学研 究 室 原 な つ み 温泉地 では、 しば しば温泉水 の流 れに沿 って微 生物 が群集塊 を形成 して生育 して い る。 こ のよ うな温泉微生物群集 内で は、構成種間 の密 な関係が存在す ると考 え られて いる。長野県 中房温泉(弱 アルカ リ硫黄泉)に は、70℃‑80℃ の温泉水 の流水 中に無 色〜灰色〜 白色の微 生物群集塊が発達 してい る。 その集塊 の主要 な構成種 は、好熱性匂 π諺cα1ε5目の硫 黄酸化細 菌(3〃 のア1乃ッ4ア09ε刀∫ゐ∫z4〃z属)で、本菌 は好気的代謝 によって硫化 水素 また は硫 黄 を酸化 し、

硫 酸イオ ンを生成す る。 また同時 に7乃θ朋odθ5πの わαc'顔〃〃2sp.やC磁 伽10アoわ伽2sp.に 近縁 な 細菌 も共存 して お り、嫌気的代謝 に よ り硫酸 イオ ンまたは硫 黄 を還元 し硫化水素 を生成 す る ことが知 られて いる。 これ らの細菌 は硫黄化合物 の循環 に必要 であ るが、酸素 の要 求性 と感 受性 といった点で相容れ ない反応で あ る。 これ らの代謝が 同時 に起 ってい る事 を示 すのは環 境 にお ける硫 黄循 環 を考 える上 で重要 であ る。 そこで本研 究で は、温泉地 か ら採取 した微 生 物 群集塊 を実験 室内で安定 に培養 ・維持す る ことを試 み、集塊 を構成 してい る細 菌の代謝 が 同時に起 こる条件 を探 る事で、環境 中に発達 してい る微生物群集塊 内での硫黄代謝系 を明 ら かにす るこ とを 目的 とした。

長野 県中房温泉 で採 取 した微 生物 群集塊 を嫌気 条件 で実験 室 に持 ち帰 った。温泉水成 分 を 模 した人工温泉水 中で嫌気 的 に培養 す る と、培養液 中 に硫化水 素の生成 が確 認で きた。 この こ とか ら集塊 中の嫌気 的代謝 による硫酸 イオ ンまた は硫 黄の還元が あった と言 える。次 に、

培養瓶 に注射針 を刺 し、注射針 の穴(孔 径0.5mm)を 通 して培養瓶 の気相 が大気 とゆっ く り交換で きるような微好気条件 で培養 した ところ、 白色集塊 で は培養 開始後 す ぐに培養液 中 の硫化水素濃度 お よび硫酸 イオ ン濃度 の上 昇が観察 された。 この上昇 は約100時 間 に渡 って 続 いた。硫化水素濃度 の上昇 か ら、 このよ うな微好気条件で も嫌気的代謝が発揮 され た こと を示 してい る。 また、硫化水素濃度 が低下せ ず硫酸 イオ ン濃度 が上昇 したのは集塊 内に存在

した硫 黄 を硫 黄酸化細菌が酸化 し硫酸 イオ ンを生成 させ、 同時 に硫 黄 を還元す る細菌が硫 化 水 素 を生成 したた めだ と予想 さ る。一方、 白色 を呈す る硫黄 の蓄積量が少 ない灰色集塊 では 培 養開始後す ぐに硫 化水素濃度の低下が観察 され、 同時 に硫 酸 イオ ン濃度 の上昇が確認 され た。48時 間後 か らは硫 化水素濃度 は検 出限界以下の低 い濃度 を保 ち、硫酸 イオ ン濃度 も大 き な変化 は見 られず一定 を保 っていた。 この結果 よ り、 白色集塊 に は持 ち込 みの硫 黄 による細 菌への電子供 与体の供 給が あった ことが支持 され、灰色の集塊 内の硫 黄循環 を維持 す る培養 を行 うに は電 子供与体 を加 える必要 があ るこ とが示 唆 された。

さらに 白色集塊 の培養 を続 ける と、硫酸 イオ ン濃度 は上昇 を続 ける ものの硫化 水素濃度 は 低下 していった。 これ は硫 黄酸化細 菌の利用 で きる硫 黄が枯 渇 し、人工温泉 中の硫 化水素 を 消費 しは じめ るの と同時に、硫酸 イオ ン/硫 黄還 元細 菌の活性 も低 下 して きた ことが考 えら れ る。

今 回実験室 内において微生物群集塊 を培養 し、集塊 中の硫酸 イオン と硫化水素 を生成 す る 細菌 の活性 を同時に検 出で きる条件 を見 いだす ことがで きた。適切 な酸素供給条件下 では、

これ らの細菌 間でそれ ぞれが行 う好気的代謝 と嫌気 的代謝 に よる硫黄化合物 の授受が進行 し て い ることを強 く示唆す る結果で あ る。

Cultivation of microbial communities from a hot spring and interaction among microbes involved in sulfur cycling

Natsumi Hara 2011

Department of Biological Sciences Graduate School of Science and Engineering

Tokyo Metropolitan University

Contents

Abstract ...

Introduction...

Materials and Methods •

Results ...

Discussion ...

References ...

Figure captions • • • •

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Abstruct

In the hot springs, microbes often forms the microbial communities along the flow of the hot spring water and they grow. It is thought that a close relation of the composition

interspecies exists in such a hot spring microbial communities. At Nakabusa hot spring (slight alkaline sulfur hot spring) located in Nagano Prefecture, clear-, gray-, and white- microbial communities develope in 70-80°C flowing water. The major component species of these communities is themophilic Aquilicales sulfur oxidizing bacteria (Sulfurihydrogenibium sp.), this bacteria produce sulfate to oxidize sulfide or elemental sulfur by aerobic metabolism. It was identified the coexistence of

Thermodesulfobacterium sp. and Caldimicrobium sp., they produce sulfide to reduce sulfate or elemental sulfur by anaerobic metabolism. These aerobic and anaerobic bacteria are neccesary for sulfur compounds cycling, however it is happend

contradictory reactions at the same time. It is important for thinking about the sulfur cycling in the environment to show that these metabolizing has happened at the same time. In this study, to clarify the sulfur cycling in microbial communities, we searched the conditions that microbes can metabolize under, in the way to maintain the stable- culture of the hot spring microbial communies in the laboratory.

The microbial communities "streamer" were collected and brought to the laboratory under anaerobic. The cultivation under anaerobic condition in artificial hot spring water, the sulfide production was observed. This result indicated the there are the reduction of

sulfate or elemental sulfur in the streamers by anaerobic metabolism. Then, Under

microaerobic condition; through the injection needle(hole diameter 0.5 mm) to the

butyle cap; the sulfide and sulfate production were observed in white color streamers.

This production continued for 100 hours. This result indicated that anaerobic metabolism was exercised even under microaerobic. It was suggested that why the increasing of sulfate without the consumption of sulfide, is the existence of elemental sulfur which can be used sulfur oxidizing and sulfur reducing bacteria. On the other hand, in clear and gray color streamers which are not contained so elemental sulfur, the the sulfide concentration was decreased immediately and the sulfate was increased at the same time. After 48 h, the sulfide concentration was kept low level and the sulfide concentration was not changed dramatically. From these results we suggested that electron donor by elemental sulfur was supplied in white color streamer, and it is need to supply the electron donor to achieve the long time stable-culture in the clear and gray color streamers.

Moreover, to continue the cultivation of white color streamers, the sulfate concentration was increased by contrast the sulfide concentration was decreased. It was supported that sulfur oxidizing bacteria consumed the sulfide because of the lack of elemental sulfur, and sulfate/sulfur reducing bacteria was lost own activities.

In this experiment, we could find the conditions that sulfur oxidizing bacteria and sulfate/sulfur reducing bacteria can active under, at the same time in the streamers.

These results indicated the transfer of sulfur compounds were happened by aerobic and

anaerobic metabolism.

Introduction

Microorganisms play an important role in the groval cycle of various elements. Sulfur is an essential nutrient for organisms and it is cycling though a variety of sulfur

compounds on earth (12). In sulfide-rich environments, some microorganisms play a major role in energy production and the maintenance of the microbial community by utilization and cycling of sulfur (20). Sulfur-rich hot spring streams provide favorable conditions for the development of microbial communities, which contain

physiologically and phylogenetically different groups of bacteria, such as chemotrophic sulfur bacteria, cyanobacteria, and phototrophic bacteria (8).

At Nakabusa hot spring in Nagano prefecture, Japan, DGGE based phylogenetic analyses of sulfur metabolizing bacteria were previously reported at different

temperatures of the microbial communities . In the hot spring, the bacterial community structure dramatically changes with temperatures at the border at 70°C, because of no phototrophic bacteria above 70°C. In this high temperature environment, microbial

`streamer' develop in hot spring water flow. This microbial streamer was dominated by Aquificae-like microbes and Thermodesulfobacteria-like microbes (23). Phylogenetic

analysis of gene for sulfur metabolizing enzymes indicated the existence of bacteria

related to Thermodesulfobacteria (7,33), and Sulfurihydrogenibium (2,4,24,28,30) in

Nakabusa hot spring (Hase,2006 unpublished).

Sulfur oxidizing bacteria

(i)Chemolithotrophic sulfide oxidation

Obligate chemolithotrophs need an inorganic source (elemental sulfur, sulfite,

thiosulfate or sulfide) for energy, and CO2 as their carbon source. Oxygen is a universal electron acceptor used by the colorless sulfide oxidizers. The important reactors

involved in chemolithotrophic oxidation of sulfide and sulfur under aerobic conditions can be summarized as:

S0 + 3/202+ H20 ---* SO42- + 2H+

H2S + 202 —p SO42- + 2H+

(ii)Sulfurihydrogenibium sp.

A taxonomic study was reported on a novel thermophilic, chemolithoautotrophic, sulfur-oxidizing microaerophilic Sulfurihydrogenibium strains isolated from terrestrial hot springs. The strains can use elemental sulfur, sulfite, thiosulfate, ferrous iron or hydrogen as electron donors, and oxygen as electron acceptor. Some strains are also able to grow anaerobically, with elemental sulfur, arsenate and ferric iron as electron acceptors. The temperature growth range was 50-80°C and mildly alkaline.

Sulfate/sulfur reducing bacteria

(i)Chemoorganotrophic sulfate/sulfur reduction

A variety of compounds could serve as electron donors and often simultaneously as

carbon sources for sulfate/sulfur reducing bacteria. In addition to sulfate and sulfur,

most species of sulfate/sulfur reducing bacteria can utilize thiosulfate and sulfite as

electron acceptors.

(ii) The phylum Thermodesulfobacteria

The phylum Thermodesulfobacteria represents one of the deeply branching lineages within the domain Bacteria and comprises rod-shaped, anaerobic, thermophilic, sulfate reducing bacteria. The group of microorganisms belonging to the genus

Thermodesulfobacterium uses lactate and pyruvate as electron donors, and sulfate or thiosulfate as electron acceptors. In recent reports, it was clarified that some genus are not capable of sulfate reduction, and these bacteria can use elemental sulfur as electron acceptors.

These bacteria use sulfur compounds as electron donors or acceptors to obtain energy.

In a previous study from our laboratory, they suggested that in the microbial streamers over 70°C, sulfur oxidizing bacteria and sulfide reducing bacteria consume and produce sulfide in situ, and they establish sulfur cycling. To clarify these suggestions, they maintained the streamers and measured sulfide concentration in vitro for 100 minutes.

This in vitro sulfide-monitoring experiments indicated slight sulfide generation by sulfate reducing bacteria under anaerobic condition, and rapid consumption of sulfide by sulfur oxidizing bacteria under aerobic conditions. However, the relationship among sulfur oxidizing bacteria and sulfate reducing bacteria in situ has not been clear yet.

In this study, to find the conditions to maintain the microbial community structure to

metabolize sulfur compounds for a long term, we tried to maintain the stable co-

cultivation system of the aerobic bacteria and the anaerobic bacteria. In order to clarify

their interspecies relationship, experiments were performed from the aspect of the

suitable air conditions and the source of nutrition for the streamers.

Materials and Methods

Sampling site

The sampling site was Nakabusa (1400 m altitude, 36°23'10"N, 137°45'00"E), a slightly alkaline and sulfide containing hot spring, located in Nagano Prefecture, Japan.

Three types of streamers were collected at the site. Details of the sampling sites are summarized in Table 1 and Fig. 1. The microbial streamer samples were kept in a sterile plastic tube (50 ml) with spring water to prevent the oxidation of samples in anaerobic bags. Spring water for inorganic analyses was immediately fixed with the same volume of 90 mM zinc acetate solution for determination of sulfide concentration. All tubes capped were transferred to the laboratory within 1 day after sampling. Spring water samples for inorganic analyses were stored at 4°C until use.

The microbial mat and streamer samples for molecular analysis were kept in 1.5 ml centrifuge tubes and then frozen at -20°C until DNA extraction.

DNA extraction

About 0.1 g of the streamer was mixed in 1.5 ml tubes with 700111 of TPM buffer (50 mM Tris-HC1 pH7, 20 mM MgCI, 1.7% polyvinylpyrrolidone K25), about 100 mg of zilconic-silicon beads (0.1 mm dia.) and about 25 mg of beads (1.0 mm dia.). Cell were lysed by shaking with a bead beater at 2,500 rpm for 1 min (MINI-BEADBEATER, FULLTECH). After centrifugation at 15,000 rpm for 2 min at 4°C, the supernatant was discarded. 7001A of phenol based lysis buffer (5 mM Tris-HC1 pH7, 5 mM EDTA, 0.1%

SDS, 6% water-sat-phenol) was added to the precipitate, then beating at 2,500 rpm for

1 min and centrifuged 15,000 rpm for 2 min. 500111 of Tris-sat-phenol was added to the supernatant, then mixed for 3 min and centrifuged 15,000 rpm for 5 min. The aqua phase was moved into a new tube, and 100111 of 5 M NaCI and 80111 of 10% CTAB/0.7 M NaC1 was added and incubated at 65°C for 20 min. 600111 of CIAA (chloroform-

isoamyl alcohol; 24:1 v/v) was added, mixed for 3 min and centrifuged 15,000 rpm for 5 min. Then the aqua phase was purified with 600111 of PCI (phenol-chloroform-isoamyl alcohol; 25:24:1 v/v/v) and 600µ1 of isopropanol. After centrifugation at 15,000 rpm for 10 min, the supermatant was discarded, and pellets were washed with 70% ethanol and then dried by vacuum. DNA was resuspended in 30p1 TE and stored at 4°C.

PCR amplification of the 16S rRNA gene

DNA fragments encoding the 16S rRNA of domain Bacteria were amplified using the

following primers: 27F2-FAM (5'-AGAGTTTGATCMTGGCTCAG-3') (3)and 907R2

(5'-CCGTCAATTCCTTTRAGTTT 3') (4). PCR amplifications were performed with

50µ1 volumes containing 1-10 ng of template DNA, 1 xEX Taq buffer (Takara Shuzo,

Kyoto, Japan), 25 mM each of MgC12, 2.5 mM each of deoxynucleoside triphosphate,

25 pmol each oh the primers, 1.25U of EX Taq DNA polymerase (Takara Shuzo, Kyoto,

Japan), and super-pure distilled water. PCR amplifications were performed with initial

denaturation at 95°C for 1 min, followed by 25 amplification cycles of 94°C for 30 sec,

52°C for 45 sec, and 72°C for 1 min, and a final extension at 72°C for 4 min. PCR

products were analyzed by electrophoresis in 1% agarose (in 1XTAE) gels containing

ethidium bromide (1 µg m1-1). PCR products were stored at 4°C until use.

PCR purification

The PCR products were purified with the QlAquick PCR Purification Kit (QIAGEN).

Restriction enzyme treatment of 16S rRNA gene fragments Amplifide fragment were digested with the restriction enzyme MspI.

Restriction digests consisted of 4p1 enzyme mixture (1111 of l Oxbeffer [TOYOBO], 1111 of 0.1% BSA[Takara], 0.5111 volumes containing 5U of restriction enzyme

MspI[TOYOBO], and 1.5111 of super-pure distilled water) and 6111 volumes containing 20ng of amplified PCR product. DNA was digested at 37°C overnight under light interception and then stored at -20°C until use.

T RFLP analysis of 16S rDNA

The lengths of the T RFs from the amplified rDNA products were determined by ABI 3730 (Applied Biosystems 3130/3130x1 Genetic Analyzer ), as follows. 0.3µ1 of

digested DNA was mixed with 151A1 of the mixture (HIDI formamide-size standard mix;

40:1 v/v). Then, this mixture was denatured at 94°C for 3 min and immediately chilled

on ice and analysed. The lengths of fluorescently labeled T RFs were determined by

comparison with internal standards by using Peak Scanner Software v. 1.0 (Applied

Biosystems).

Analytical methods

In situ temperature and pH of hot spring water were measured with the electrode method using a thermometer (Check tempi by HANNA) and a pH sensor (Twin pH, AS ONE). Sulfide concentration was measured colorimetrically by the methylene blue formation method (Cline, 1969). Sulfate concentrations were analyzed by ion chromatography (LC-20AD SP, Liquid Chromatograph, SHIMADZU, Japan) and determined by using Chromatpac (SHIMADZU, Japan).

Cultivation conditions

An artificial hot spring water was prepared to simulated Nakabusa hot spring water.

The concentrations of sulfide, SO42-, Cl-, PO42-, and CO32- in the artificial hot spring water for cultivation were, 0.3 mM, 0.5 mM, 1 mM, 1 mM, and 1 respectively (Table 2). The microbial streamers were collected at Nakabusa hot spring and kept in hot spring water at room temperature until use. 2.0 g(wet weight) of the microbial streamers were incubated in 100 ml of artificial hot spring water in glass vial at 70°C with

reciprocal shaking (100 rpm). Air phases ware set up for anaerobic, aerobic, and

microaerobic conditions. Anaerobic condition was achieved by replacing the gas phase

with N2 gas and the vial was capped with a butyl rubber and an aluminium cap. Aerobic

condition was achieved by the air phase and the vial was capped by butyl rubber and

aluminium cap. For the microaerobic condition, the air phase was replaced with N2 gas

and capped, then the cap was stuck with a 00.5 x25 mm injection needle.

Results

Characteristics of microbial communities over 70°C

The T-RFLP fingerprintings of bacteria 16S rRNA gene fragments revealed 4 termina restriction fragments ^^^ from 6^-7^^C streamer samples. ^ tech basis ofDNA sequences detected from Nakabusa hot spring, T-RFs with sizes of 95 and 270 would be derived from ^u^^ nibium azorense, that of 117 would be derived from

Thermus kawarayensis, and that of 154 would be derived from Caldimicrobium sp..

Three types of streamer were collected in 17 November 2010. From T-RFLP analysis, relative abundance of]6^r^7^/\ gene fragments of different types uf the streamer was revealed as shown in Fig. 2. The T-RFs with the sizes of 95, 270, and 117 were present in all samples. At this time, the T-RF of 154 was not detected in the clear streamer. ^^

FlFo^und27O were present in most of the streamers. In white streamer, sum of the

peak at 95 and 270 bp account for 69% in the total, 117 bp 4%, and 154 bp 6%. In clear streamer, the bands of 95 and 270 bp account for 86% in the total, and 117 bp 2%. In the gray streamer, the bands of 95 and 270 bp account for 48% in the total, 117 bp 5%, and l54bp0Y6.^^Hi^^ derived from sulfate reducing bacteria ^^enno^^un^^boc/^r/um sp. was not detected in this experiments. These streamers developed the different

sulfide concentration areas. White color streamer was observed in lower level sulfide concentration area than gray. And the difference was observed by the sampling date in the sulfide concentration. To be winter, to be higher level concentration in all areas.

Then, microscopic observation indicated that white color streamer contained a lot of

sulfur globules in the streamer shown as Fig. 3.

Detection of sulfide producing ability

Nakabusa hot spring was a slightly alkaline, sulfide-containing water. In this experiment artificial hot spring water like real Nakabusa hot spring water was used.

Consumption and production of sulfide by the white color streamer in artificial hot spring are shown in Fig. 4. Under anaerobic conditions, the sulfide concentration increased immediately from the beginning of incubation, and after 336 h incubation gradually increased to 250 11M. On the other hand, under initially aerobic conditions, the sulfide concentration decreased below detection limit within 1 h after incubation and did not increase. Supplemental addition of 1 mM of lactic acid, 1 mM of Mg2+, 3 mM of N H4+, and trace elements, in to the artificial hot spring water did not affect the results under anaerobic condition (Fig. 5). Under initially aerobic conditions, the sulfide

concentration decreased below detection limit as observed in Fig. 5; however the sulfide concenration increased after 200 h of incubation.

02 effects during sulfide producing period

After 400 h cultivation under initially anaerobic and aerobic conditions, 02 was supplied through an injection needle. The sulfide concentration change was shown in

Fig. 6, and arrows indicate the time of injection. Under initially aerobic conditions, the sulfide production was observed after 330 h incubation. Supplementation of 02 at the time decreased the sulfide concentration. Microscopic observation after 500 h

incubation indicated the existence of a large number of long rod cells (over 101..tm long) which could be Sulfurihydrogenibium sp.. Under anaerobic conditions, the sulfide concentration was increased to 27511M until 400 h. After the supplementation of 02,

sulfide concentration decreased in two samples tested. After 500 h incubation, change of

the sulfide concentration were different between different samples. When there are more large shape bacteria like SOB (Fig. 6; 2-b) in the microscopic observation, the sulfide concentration was decreased. When there are more small shape bacteria like SRB (Fig.

6 ; 2-c), the sulfide concentration was transiently decreased by 02 supplementation.

These results suggested that the number of bacteria in microbial community effected the sulfide production under microaerobic condition.

Sulfide production and consumption under microaerobic conditions

Change in sulfide concentration under microaerobic conditions was shown in Fig.7.

Initial concentration of sulfide in the artificial hot spring water was 256 laM. The sulfide concentration was decreased at the beginning of incubation with 25 h, then increased.

Consumption of sulfide concentration was observed again after 75 h incubation. After that, small increase and decrease in sulfide concentration ware observed.

The relationship between Sulfide and Sulfate concentration

At Nakabusa hot spring over 70°C, microbial communities of three color types; white, clear and gray, were observed.

The sulfide and sulfate concentrations in 2 weeks cultivation of white streamer were

shown in Fig. 8. Changes of under anaerobic conditions, the sulfide concentration was

increased for 24 h and kept around 1.3 mM until 275 h. The sulfate concentration was

increased until 48 h, after that it was decreased. Under microaerobic conditions, the

sulfide concentration was increased to 1.3 mM until 48 h, then after 140 h cultivation it

was decreased. The sulfate concentration was increased from 1.5 mM to 6.0 mM for 275 h cultivation.

The sulfide and sulfate concentration for 2 weeks cultivation of the clear streamer was

shown in Fig. 9. Under anaerobic conditions, the sulfide concentration was increased

between 25 h and 100 h after the start of incubation, then kept 1.3 mM until 275 h. The

sulfate concentration was kept about 1.5 mM for 48 h cultivation, after that it was

decreased and kept 0.8 mM until 275 h. Under microaerobic conditions, the sulfide

concentration was decreased immediately below detection limit. The sulfide and sulfate

concentrations for 2 weeks cultivation of gray streamer was shown in Fig.10. Under

anaerobic conditions, the sulfide concentration was increased to 1.3 mM until 48 h and

kept its concentration until 275 h. The sulfate concentration was decreased after 24 h

cultivation, and it was kept at 0.8 mM until 275 h. Under microaerobic conditions, it

was the same tendency as the results in the clear streamer.

Discussion

I found conditions for maintaining active microbial communities from natural habitat with sulfur oxidizing bacteria and sulfate/sulfur reducing bacteria in vitro for several days. Microbial streamers in Nakabusa hot spring over 70°C were dominated by sulfur oxidizing bacteria (Sulfurihydrogenibium sp.) and sulfate/sulfur reducing bacteria (Thermodesulfobacterium sp., Caldimicrobium sp.). Sulfur oxidizing bacteria (SOB) can use elemental sulfur and sulfur compounds as electron donors, and oxygen as electron acceptor (2,24,28,30). On the other hand, sulfate/sulfur reducing bacteria (SRB) can use sulfate or elemental sulfur as electron acceptors, and some organic compounds as electron donors (19,11,29). Sulfurihydrogenibium sp. is microaerophilic bacteria, and Thermodesulfobacterium sp. and Caldimicrobium sp. are anaerophilic bacteria. Therefore, it might be happened both aerobic and anaerobic metabolisms are present at the same time in the microbial community. To clarify the ways to metabolize sulfur under such different conditions, we tried to find the conditions to maintain the microbial community with the native form of streamer, and searched the conditions that ware appropriate for growth of both bacteria in the in vitro communities.

Microbial composition and sulfur globules

In the samples around 70°C at Nakabusa hot spring, it was found that there were three main species; Sulfurihydrogenibium sp., Thermus sp., and Caldimicrobium sp.

Sulfurihydrogenibium sp. and Thermus sp. (3,14) known as sulfur oxidizing bacteria,

and Caldimicrobium sp. is a species of sulfur reducing bacteria. From the results of T-

RFLP in Fig. 2, the major components in all streamers investigated were sulfur oxidizing bacteria. By comparing between white and gray color streamers, it was

formed that the white streamer had more SOB than the gray one, on the other hands, the gray streamer had more SRB than the white one.

In a previous report (Hase,2006 master degree thesis, Tokyo Metropolitan University), DGGE results indicated the presence of Thermodesulfobacterium sp. However, in the T RFLP analysis, the T RF of Thermodesulfobacterium sp. was not observed in this study.

Anaerobic cultivation

Sulfurihydrogenibium sp. is a microaerophilic bacterium and Thermodesulfobacterium sp. and Caldiicrobium sp. are anaerophilic bacteria. Air conditions are important factors to maintain all those bacteria. Under anaerobic conditions, all samples produced sulfide

and kept high level sulfide concentration after about 150 hours of cultivation. It might be caused by sulfate/sulfur reducing bacteria (SRB), such as Thermodesulfobacterium sp. and Caldimicrobium sp. By comparing between white and gray color streamers (Fig.

8, 10(A)), white streamer produced sulfide faster than the gray one. It suggested that there were many sulfur globules in white color streamers, and therefore Caldimicrobium sp. may be used elemental sulfur as electron acceptor and produced sulfide. On the other hand, sulfate production was detected only in white streamer even if 02 was not present. It was reported that some anaerobic bacteria are able to catalyze the

disproportionation of elemental sulfur to sulfate and sulfide (10,16). Therefore, I

suggested that Caldimicrobium sp. produced sulfate by using elemental sulfur which

was contained in white streamer by the disproportionation.

Aerobic conditions

From Fig. 4 and 5, under aerobic conditions, by the addition of Mg2+, NH4+, lactic acid and trace element, sulfide production was detected after 200 hours of cultivation. An isolation medium of Sulfurihydrogenibium sp. contains magnesium and nitrogen source in the forms of Mg2+ and NH4+ . From the result of sulfide producing, I can suggest that initial sulfide was oxidized by sulfur oxidizing bacteria (SOB), such as

Sulfurihydrogenibium sp. by 02. This condition would be suitable for SOB, which consumed 02 as electron acceptors withi 200 hours after the start of cultivation, and then made the air phase anaerobic. After that, SRB in the streamer stayed alive, produced sulfide because of suitable anaerobic condition for SRB after the 02 consumption.

These results indicate that the supplements, Mg2+ and NH4+, improved the growth of SOB, resulted in the sulfide production in the streamer. Therefore, I suggested that supplements(Mg2+, NH4+, lactic acid and trace element) were need to maintain the SOB and SRB activities.

From Fig. 6, I suggested that the community structure in the streamer was changed during 400 hours cultivation, and this change affected the sulfide production. In the streamer which has more SOB (Fig. 6:2-a, 2-b), supplemented 02 and sulfide were consumed immediately by SOB. On the other hand, in the SRB dominant streamer (Fig.

6:2-c), the effect of supplemental 02 was transient on the sulfide concentration. These

results suggested that the community structure and the present of 02 were important for

maintaining the sulfur cycling in the streamer.

Microaerobic conditions

Fig. 7 shows that microaerobic condition was important for forming the microbial community containing both aerophilic and anaerophilic bacteria. This condition would make both aerobic and anaerobic parts in the streamer. Therefore we were able to observe the sulfide production and consumption due to the sulfate oxidizing and sulfide reducing activities by SOB and SRB. Changes of sulfate and sulfide concentrations

indicated the presence of the sulfur cycling in the streamer in vitro. Fig. 8, 9 and 10(B), show the sulfur cycling in white, clear and gray color streamer, respectively under the microaerobic conditions. All three streamers produced sulfate in 48 hours. However after 48 hours, only the white color streamer continued to produce sulfate,

accompanying the production of sulfide. I suggested that elemental sulfur was used by SOB as an electron donor and SRB as an electron acceptor. Microscopic observation indicated that the white color streamer had more elemental sulfur in the form of sulfur globules within the microbial community. Moreover, it was reported that some sulfur oxidizing bacterial species can store sulfur intracellular as an electron donor and oxidize

it (17,27,31). SOB in the white color streamer stored sulfur in the natural habitat and oxidized it to sulfate in this cultivation system. It is not clear in Sulfurihydroganibiur

sp. of this system yet, but some thermophilic sulfur bacteria have the system that oxidize intracellular sulfur (5,26). At the same time, Caldimicrobium sp. produced

sulfide by reducing elemental sulfur. The other streamers might contain sulfide too,

however there were less amount of elemental sulfur to be able to be used by

Caldimicrobium sp. During the additional 150 hour cultivation, the sulfide was

consumed in the white color streamer, and sulfate was produced continuously. This result suggested that SOB depleted with intracellular sulfur first and then begun to oxidize sulfide. It might be one of the reasons why there were originally a lot of SOB in the white color streamer than the gray color streamer. In natural environment, the gray color streamer would keep its community structure by continuous supply of sulfide from the spring water. During the long time cultivation such as about 300 hours, under microaerobic conditions, sulfide production did not continue because of the

consumption and lack of electron donors as organic compounds.

In natural environment, microbial communities developed with interaction among constitutive species. Over 70°C, chemotrophic bacteria interact through sulfur cycling to oxidize and reduce sulfur compounds. A model of microbial sulfur cycling in this part of the streamer in Nakabusa hot spring is shown in Fig. 11. To metabolize sulfur

compounds properly, air conditions and supply of electron donors are very important.

In this study, we showed that the external microaerobic conditions were a major factor to maintain both SOB and SRB activities. Under the flowing water, the streamer structure would make the outside of the streamer microaerobic and inside anaerobic.

However, even if the air phase was suitable, both activities did not continue because of

the lack of electron donors. At low sulfide concentration, SOB accumulated elemental

sulfur by oxidizing of sulfide. Therefore, it may be the reason why we could observe

three types of streamers at the same temperature with the same dominant species. The

electron donor's continuous supply is necessary to achieve the long term cultivation

more than this study period.

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Acknowledgements

I especially grateful to K. Matsuura and S. Haruta for their advice and valuable

discussion. I would like to thank S. Nagashima and S. Hanada for thier suggestion. I

thank to H. Otaki and K. Yamamoto for their help and technical suggestions. Finally, I

thank to Environmental Microbiology laboratory members for their strong support.

Tablel. Characteristics of spring water under which three kinds of streamers

ware collected at Nakabusa hot spring

Sample

Temp. (°C)

Sulfide concentration(iiM)

White color streamer

(I) (II) (III) (IV) (VI) Clear streamer

(I) (II) (III) (IV) (VI) Gray color streamer

(I) (II) (III) (IV) (VI)

76 70 75.3 79.3 70

65

71 69.8

71

70 74.3

71 78.1

73

6.9 6.8 7.5 7.2 7

8.5 8.2 8.5 8.7 8.3

6.8 8.4 8.7 8.7 8.4

39.2 79.1 122.9 156.1 243.1

53.8 108.5

150 121.6 429.2

333.2 427.4 303.4 182.5

(I) (IV)

12 June 2010, (II ); 10 July 2010, (III); 19 August 2010, 29 October 2010, (VI); 17 November 2010

no tested

Table 2. Composition of artificial hot spring water.

1 mM NaCI

1 mM NaH2PO4 - 2H20 0.5 mM Na2SO4

(Sulfur source)

0.3 mM Na2S • 9H20 1 mM NaHCO3

Adjusted at pH 8.0

.--,

(A)

.;"4 ,;;c•..

•,;.; ,

if

J:::',- '` ..'' .;

. ,

:1

a i4 I., ...“ '

lirtYY • , ... 1 -'t r'

t :);, ,ie,'.• . '''.h. w

. ! • • ,... ..,-,.,,,,,„ , r : ;•',.,—.41..L.t: •;,,,, °

t, cp..;.7,,,,,..:,,..- •,1-.J

...i.,,,- .4..:.•

(C)

Fig. 1. Photographs of the streamer at Nakabusa hot spring.

Arrows respectively indicated White color streamer(A), Gray color streamer(B), and Clear streamer (C).

The dashed circles in small photo were sampling sites of gray color streamer and clear streamer.

32

80 ---

T-RFs(bp)

^95

60 - • 270 ~

117

o• 154

a) U

a40 -

-0 C D

C 20 -

IF cu

Tb cc 0 II II

White color streamer Clear streamer Gray color streamer

Fig. 2. Relative abundance of 16S rRNA gene fragments retrieved from three

types of the microbial communities streamers on T RFLP analysis. The T RFs of

95(blue bar) and 270 (green bar) represented Sulfurihydrogenibium sp., 117(yellow

bar) represented Thermus sp., and 154(red bar) represented Caldimicrobium sp.

(a)

mum

(b)

Fig. 3. Microscopic images of microbial streamers. (a);Images of white color

streamer containing sulfur globules. (b);Images of gray color streamer.

300 ---

225 -

c 150 0 CD

75

cn

0 100 200 300 400

Time ( hour )

Fig. 4. Sulfide production and consumption by the white color streamer in artificial hot spring water. •(circle), the sulfide concentration under anaerobic cultivation. ^ ( diamond ), the sulfide concentration under aerobic cultivation.

35

300 ---

225 - C

c 150 -

O 0

0

75 - cn

0

0 100 200 300400

Time (hour )

Fig. 5. Sulfide production and consumption with the white color streamer in artificial hot spring water. .(circle), the sulfide concentration under anaerobic cultivation. • ( diamond ), the sulfide concentration under initially aerobic cultivation.

36

2 4

C 0 c 0 0

E cn

300

225

150

75

0

0 100 200 300 400 500

Time (hour )

300

225 c U

0 150

0 01 mi 75 N

0

0 100 200 300 400 500

time (hour )

2 4

0 C 0 0 m E c U)

300

225

150

75

0

0 100 200 300 400 500

time (hour )

Fig. 6. Effect of Oz supplementation changes of the sulfide concentration after supplementation of 02.

Air was supplied through an injection needle after the time indicated by arrow in graph(').

(1-a) Sulfide production and consumption under initially aerobic condition. Microscopic observation after 475 h cultivation was indicated (2-a).

(1-b,c) Sulfide production and consumption of different two samples under initially anaerobic condition. Microscopic observation after 475 h cultivation was indicated (2-b,c).

White solid arrow indicates long rod cells (2-a,b).

500

i 375

U c 250 O V N

to 125

0 100 200 300 400

Time ( hour )

Fig. 7. Sulfide production and consumption with the white color streamer under microaerobic conditions.

38

2.5 I- 2.5

ff

2.0- 2.0 E

UC

O 1.5. 1.5c

O0

0

1.0 -1.0

4- c 0

.5 -. 0.5 (1)

0--- 0

0 75 150 225 300

Time(hour)

9.0

(B)--- 9.0

7.5 --7 .5

g 6.0 --6 .0 E

o4.5 --4.50

733.0 --3.0

=4-co

u 1

.5 •.1.5 w

00

0 75 150 225 300

Time(hour)

Fig. 8. Sulfide and sulfate concentration with "white color" streamer for long term cultivation under anaerobic condition(A), and microaerobic condition(B) .

•(circle),data for the sulfide concentration. , the sulfate concentration .

39

3.0 2.5 2

g 2.0

E U

o 1.5

U N 1.0 U) 0

.5 0

3.0 2.5 2

E 2.0

U

0 1.5

U a) 1.0 n U)

0.5 0

0 75

150

Time(hour)

225

300

0 75 150

Time(hour)

225 300

3.0 2.5

2.0 g

0

1.5 0

0

1.0 1 0.5w

0

3.0 2.5 2.0 1.5 1.0

E

0 C 0 0 a) 4--(II

0.5 u) 0

Fig. 9. Sulfide and sulfate concentration with " clear" streamer for long term cultivation under anaerobic condition(A), and microaerobic condition(B).

•(circic), the sulfide concentration. , the sulfate concentration .

3.0 2.5 2

2.0 E U

o 1.5

U a)

0 1.0

(f) 0.5

0

3.0 2.5 2

2.0 E U

0 1.5

U N

0 1.0

Cl) 0.5

0

0

75 150

Time(hour)

225 300

0 75 150

Thime(hour)

225 300

3.0 2.5

2.0 g

0

1.5 0

0

1.0 0.5U'

0

3.0 2.5

2.0 E

0

1.5 0

U

1.0 %

4-

0.5 (I) 0

Fig. 10. Sulfide and sulfate concentration with " gray color" streamer for long term cultivation under anaerobic condition(A), and microaerobic condition(B).

•(circle), the sulfide concentration. , the sulfate concentration .

H2S

S2-

So

lfurihydrogenibiun'i sp.

Thermodesulfobacterium sp.

imicrobium sp .

So - -

41 ...

.

^

..,

SO42-

,

4

oxic

anoxic

Fig. 11. The schematic model of cross-section view of sulfur cycling

in the streamer over 70°C in Nakabusa hot spring.