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 +3  3 ῌ῎Relation between Bacteria and Deposits found in the HighPCO, Spring Waters of Primorye, Far East RussiaKenji SUGIMORI+, Oleg CHUDAEV,, Valentina CHUDAEVA-, Hiroaki IGARASHI.,
Akihito KUNO/, Motoyuki MATSUO/ and Yousuke SUGIMORI0(Received Mar. +3, ,**1, Accepted Jun. 1, ,**1)
Department of Biology, Toho University School of Medicine, Tokyo +.-ῌ2/.*, JapanFar East Geological Institute, Russian Academy of Sciences, Vladivostok 03**,,, Russia
Paciﬁc Institute of Geography, Russian Academy of Sciences, Vladivostok 03**.+, Russia.
Department of Anatomy, Toho University School of Medicine, Tokyo +.-ῌ2/.*, Japan/Graduate School of Arts and Sciences, The University of Tokyo, Tokyo +/-ῌ23*,, Japan0
Department of Food Science and Technology, National Fisheries University,Yamaguchi 1/3ῌ0/-/, Japan+,ῌ

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U82  !"#$%&' ()*+,-./0123 3' 45267892Acidithiobacillus ferrooxidans 2:;3&2<=  >&' ?@AB
' CD3 EFGHIJKLMNOPQR3 S' TUVWXYZ[' EFG\]^_`abGcd92ef& >&' gEFG)7h3 \]ij Bkl3 AEFG2mnij%& o>' CGklpqrstEFG7h
[B' kl3&uvw[OPAEFG7h
2x%& EFG7hrstyx zA{' WCGkl3 7h2|I[% }~&00 : ' EFG'  ' 0,' _`abG'  2 \]Abstract
The Tiga forest of Primorye is located in the Vladivostok and Khabarovsk regions ofthe Far East Russia. There are more than +** springs with either Ca-HCO- or Na-HCO-type waters, and these contain high levels of PCO,, ferrous ion and a variety of rare elements. Electron microscopic observation of deposits from these springs revealed thatthey were mostly formed by inorganic matter, and that they had a core of inorganic ororganic material. Occasionally, bacterial organisms were found in the deposits. Cultivation of these spring waters with deposits showed a few bacterial growth in inorganic
media with ferrous ion. According to these bacterial cultivated conditions using ferrous
ion for their growth and their short-rod forms, these bacteria resembled Acidithiobacillus
ferooxidans, but could not be identiﬁed precisely because of their di$cult cultivation.
After a few months’ cultivation, deposits were formed with strongly adhered to the glass
tube wall ; the deposits were di$cult to dislodge with gentle tapping. The presence ofweb-like structures with octahedrons crystals in the cultivated deposits may explain thedurability of the deposits ; these networks were not present in the control medium. Theseﬁndings show that the formation of deposits in spring is strongly related to the presenceof microorganisms such as bacteria.
Key words : bacteria, deposits, high PCO, spring, Primorye, string-like structure, octahedrons crystals+.

Introduction

The Tiga forest of Primorye is ,/*,*** km, in area, located in the Vladivostok and Khabarovsk regions in Far East Russia. This area is well-known for its abundant deposits of rareelements. More than +** springs are located in the Primorye Area (Chudaev +332ῌ+333). Mostof the springs are cold, and only , springs located near the Japan Sea side, can be called hotsprings”. These springs have typically a high PCO, (up to ,.0 atm) and a high concentrationof Ca, and HCO-, and are also important for Na, Mg, or K (Shand et al. +33/, Chudaeva
et al. +333, Chudaev et al. ,**+). In addition, each spring contains a variety of rare elements(Shand et al. +33/, Chudaeva et al. +33/ and +333, Chudaev et al. ,**+). Fe ions are also high(Shand et al. +33/, Chudaeva et al. +333, Chudaev et al. ,**+), and most of these springs areorange due to ferrous deposits that had been oxidized from Fe, to Fe-.
The chemical composition of the water of the high PCO, Schmakovka Spring, near Lake
Khanka, was investigated in the +3th century. This work was performed by the Far East /1  ,**1

83

Geological Survey, the Central Institute of Health and Physiology, and the Primorye Territorial Geological Survey. Most of the data from these projects were not published and are kept
in the archives of the Far East Geological Survey and Primorye Territorial Geological Survey.
After these studies, the springs were reevaluated for their use in energy production ; then, theheat sources of the Chistovodnoe Area and the possibility of mine deposits in the Lastochika
and Gornovodnoe Areas were researched (Ushakin ed. +303). Recently, Chudaeva and her teaminvestigated the chemical composition of these springs, particularly those near VladivostokCity, and published the book, Mineral Waters of Primorye (Chudaeva et al. +333). Their subsequent researches were continued, focusing on the springs of Primorye, and new geochemical
data were presented (Chudaev et al. ,**+). Most of these data were published in Russian, butsome of the ﬁndings concerning the high PCO, spring water in Primorye have been presented
in Japanese (Sugimori ,**-).
These springs are classiﬁed as either Ca-HCO- or Na-HCO- springs. The chemical composition of these springs is characterized by the frequent presence of high concentrations ofrare elements, especially Cu, Ga, and Ge, and by a high PCO, (Shand et al. +33/, Chudaeva et al.
+333, Chudaev et al. ,**+, Sugimori ,**-). In addition, the concentration of Fe is also high,resulting in red deposits. Although a great deal of geochemical and geological analyse of
these springs have been done only a few studies have been reported on the microbiologicalproperties. The purpose of the present study is to investigate the biological properties forthese high PCO, springs.

,.
,. +Sampling ﬁeld and methods

Sampling ﬁeldThe ﬁelds of this study were in the Primorye Area, which is north of Vladivostok City, inthe Far East Russia. Ten high PCO, spring waters with red deposits were sampled for bacteriological analysis in Medveji, Abdeevsky, Nerobinsky, Bolshoi Kluch, Sodovy, Narzany,Lenino-,, Luzky (, samples) and Gornovodnoe. The pH and temperature of these springs weremeasured in the ﬁeld with a handy pH meter (YOKOKAWA pH2+) (Sugimori, ,**-).
,. ,Electron microscopic observation of deposits in springs
Scanning electron microscopy (SEM, HITACHI ; S./*) was used to observe deposits inwater samples. Deposits were washed with *.+M PBS and ﬁxed with ,./῍ glutalaldehyde(in *.+M PBS). The ﬁxed sample was placed on the thin cover glass (slide) coated with *.,῍
poly-L-lysine, and viewed after staining with +῍ osmic acid, and conductive staining with ,῍tannic acid and +῍ osmic acid.
,. -Biological analyse of deposits
The deposits were cultured in 3K Medium, containing K,HPO. : *./ g, (NH.),SO. : -.* g, KCl :*.+ g, MgSO.ῌ1H,O : *./ g, FeSO.ῌ1H,O : /*.* g, Ca (NO-), : +*.* mg, +*N H,SO. : +.* ml, and distilled water : +.* liter. The pH of this medium was adjusted to -.*, however it was actually
little bit higher than -.*, but no higher than ..*. The reason for this is iron oxidizing bacteria

῍ῐ῎῏

84

can not be cultured in a medium over pH .. After sterilization, +*ml of this medium wasplaced in each sterilized tube. Samples from these springs were inoculated into each tube
with the medium, to a volume of +*ῌ, and incubated at -*ῌ.
After several weeks culture, bacterial growths and the characteristics of deposits wereobserved by using a light microscope (OLYMPUS BX/*).

-.

Results and Discussion

Ten springs containing deposits were sampled in July and August +333. The spring deposits were all reddish-brown. The pH and temperature of these springs are shown in Table +(Sugimori, ,**-). These spring waters were cold and the pH was neutral to acidic. The otherdata regarding DO, HCO-, EC, and chemical components are described in Shand et al. (+33/),
Chudaeva et al. (+333 in Russsian), Chudaev et al. (,**+) and Sugimori (,**-, in Japanese).
Electron microscopic images of the spring deposits are shown in Photos +ῌ+ to +ῌ/. Photo+ῌ+ conﬁrms that the deposits were almost entirely formed of inorganic matter. Sometimes,
the deposits exhibited linearity, as shown in Photo +ῌ,. Photo +ῌ, shows inorganic matter
adhering in a regular pattern to the surface of other lining materials. Rod shaped bacteria
were found in the inorganic deposits, as shown in Photos +ῌ- and +ῌ.. A bacterium Spirochetewas also found in the deposits (Photo +ῌ/). The genus name of spices may be Leptospira with
its typical shape of spiral, which is commonly found in natural sources contaminated withanimal feces. Previous reports suggested that biological processes were responsible for thegrowth of these spring deposits (Konhauser et al. +333, Sugimori et al. +33+).
After cultivating these spring waters with deposits at -*ῌ, small numbers of bacteria

Table + Temperature and pH of the high PCO, spring waters in the SouthPrimorye, Far East Russia (+333 samples, Sugimori ,**-)Locality
SHIMAKOVKA Group+. Medveji,. Abdeevsky
SHETUKHINSKAYA Group-. Nerobinsky.. Bolshoi Kluch
SAMARUKA Group/. Sodovy
LENINSKOE Group
0. Narzany
1. Lenino-,
CHUGUEVKA Group
2. Luzky(borehole)
GORNOVODNOE Group3. Gornovodnoe

Temperature (ῌ)

pH

++4.
+*4+

040.
04/*

14.
24*/41,04/-

24,/40.

/40
242

042.4.,++42+04/040,040,

34-0411 /1  ,**1



85

Photo + Electron microscopic images of spring deposits from Primorye, Russia+ῌ+ Sodovy Spring deposits (Bar, mm)+ῌ, Bolshoi Kluch Spring deposits (Bar, mm)+ῌ- Sodovy Spring deposits (Bar, mm)+ῌ. Narzany Spring deposits (Bar+ mm)+ῌ/ Bolshoi Kluch Spring deposits (Bar+ mm)

were found in the culturees as showing an arrow (Photo -ῌ-). Bacteria capable of growing ininorganic medium by using ferrous ion for energy were found in all spring water samples
from Primorye, but only one or two short-rod cells were found in about +* ﬁelds under lightmicroscopic observation. These bacteria were cultured for 1 additional years, with - or . inoculations a year, under inorganic medium conditions. These bacteria were grown in themedium with ferrous ion for one of the energy source for growth. According from these fact,
the bacterium seems to be Acidithiobacillus ferooxidans, but not yet be identiﬁed precisely,
because of the di$cult cultivation. When the culture tubes were observed after a few months
cultivation, cultured deposits were ﬁxed on the glass tube wall, as shown in Photos ,ῌ+ and,ῌ,. These deposits strongly adhered to the glass tube wall and maintained intact after gently
shaking and tapping the test tube. However, the control tube deposits (uncultured medium,without bacteria) readily disintegrated after gentle shaking or tapping, as shown in Photo,ῌ-. After one or two months’ cultivation, light microscopic observation show that crystals
were found in the cultivated medium and in control medium (Photo -). Crystals from cultured
and control tubes were right octahedrons, and the crystals in control tube (Photo -ῌ,) wereusually smaller than the crystals in cultured tube (Photo -ῌ+). Thin string-like structures῎ῑ῏ῐ86

Photo , Cultured samples with deposits from Primorye, Russia anduncultured sample with deposits in test tubes,ῌ+ and , ῌ, : cultured samples, ,ῌ- : uncultured sample

Photo - Light microscopic observation of crystals in cultured and uncultured samples-ῌ+ Crystals formed from a cultured sample (ῌ.**)-ῌ, Crystals formed from an uncultured sample (ῌ.**)-ῌ- Thin ﬁlaments and bacteria from a cultured sample (ῌ+***)-ῌ. Spider web structure linking octahedral crystals from a cultured sample (ῌ+***)(Bar῍+* mm)were observed in cultivated deposits (Photos -ῌ- and -ῌ.). These structures resembled aspider web, with a network among crystals, and they may be formed by bacterial activity.
These networks were not observed in the control medium. These may explain why more
resilient deposits were formed in the cultured medium. We can summarized the ﬁndings inprevious papers about the relationships between microorganisms and deposits, as follows ;+. Many kinds of microorganisms were found in the spring deposits, consisted algae mainlyof found in the center of deposits and formed the deposits (Sugimori et al. +33+). ,. Some
bacteria, cyanobacteria, were found in the silica sinter by electron microscopic observation,and their cytoplasms were replaced by silica, presumably by lysed process in-situ (Konhauseret al. +333). And one of the cyanobacteria, Calothrix sp., were isolated from the surface of silica
sinter, and cultured (Konhauser et al. +333). So, the existence of cyanobacteria was so impor- /1  ,**1

87

tant for the siliciﬁcation at the hot spring in Iceland (Konhauser et al. +333). -. It was revealed
that iron and silica were accumulated inside of bacteria as biomineralization at Yellowstone
National Park (Ferris et al. +320).
These reports and our ﬁndings show that the formation of deposits in spring is strongly
correlated with the presence of microorganisms.
References
Chudaeva, V.A., Lutsenko, T.N., Chudaev, O.V., Chelnokov, A.N., Edmunds, W.M. and Shand, P.
(+33/) : Thermal waters of Primorye region, Eastern Russia. Proceedings of Water RockInteraction 2th (by Kharaka, Y. and Chudaev O.V. ed.), -1/ῌ-12, Balkema, Rotterdam.
Hudaeva (Chudaeva), V.A., Hudaev, O.V., Helnokov, A.N., Zdmunds, U.M., Wana, P. (+333) :Mineral;iye Vody Primor;q (Mineral Waters of Primorye). +1, p, Dal=naukaῌ
Vladivostok.
Chudaev, O.V. (+332ῌ+333) : Mineral waters of Primorye. Printed in Kongju University of South
Korea in +332ῌ+333 (short report with . pages).
Chudaev, O.V., Chudaeva, V.A., Sugimori, K., Nagao, K., Takano, B., Matsuo, M., Kuno, A. andKusakabe, M. (,**+) : New geochemical data of the high PCO, waters of Primorye (FarEast Russia). Proceedings of Water Rock Interaction +*th (by Cidu, R. ed.), Vol. +, .1-ῌ.10,Balkema, Lissa.
Ferris, F.G., Beveridge, T. J. and Fyfe, W.S. (+320) : Iron-silica crystallite nucleation by bacteria
in a geothermal sediment. Nature, -,*, 0*3ῌ0++.
Konhauser, K.O., phonenix, V.R., Bottrell, S.H., Adams, D.G. and Head, I.M. (+333) : Microbial
silica interaction in modern hot spring sinter. P. ,0-ῌ,00, Gheochemistry of the Earth’sSurface (Armannsson ed.), A.A. Balkema, Rotterdam.
Shand, P., Edmunds, W.M., Chudaeva, V.A., Lutsenko, T.N., Chudaev, O.V. and Chelnokov, A.
N. (+33/) : High PCO, cold springs of the Primorye region, Eastern Russia. Proceedingsof Water Rock Interaction 2th (by Kharaka, Y. and Chudaev O.V. ed.), -3-ῌ-30, Balkema,Rotterdam.
Sugimori, K., Mifune, M. and Yuhara, K. (+33+) : Biological studies of spring deposits situatedin Kuroiwa Spring, Kyushu. J. Balneol. Soc. Jap. .+ (-), +03ῌ+11.
Sugimori, K. (,**-) : High PCO, spring water in the Primorye, Far East Russia (in Japanese) J.
Balneol. Soc. Jap. /- (,), .2ῌ0*.
Ushakin, E.P. ed. (+303) : Primorskij kraj (Primorye Teritory) Gidrogeologiq SSSR (Hydrogeology of USSR, vol. XXV), /,* p, M. Nedra, Moskva (Moscow)