Title THERMOPHILES COLLECTED IN THE OKUHIDA SPA AREA
Author(s) YAMAGATA, Shuzo; HATTORI, Manabu; NAGASE, Masahiro
Citation [岐阜大学教養部研究報告] vol.[23] p.[39]-[45]
Issue Date 1987
Rights
Version
岐阜大学教養部 (Department of Biology, Faculty of General Education, Gifu University) / 岐阜大学教養部 (Department of Biology, Faculty of General Education, Gifu University) / 岐阜大 学教養部 (Department of Biology, Faculty of General Education, Gifu University)
URL http://hdl.handle.net/20.500.12099/47612
※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
DI ST RI BU T I ON OF T H ERM OPH I LI C BA CT ERI A
I N GI FU PRE FE CT U R E : I . A PRE L I M I N A RY RE PORTON T H ERM OPH I L ES COL LECT ED
I N T H E OK U H I DA SPA A RE AShuzo Y A M A GA T A , M anabu H A T T ORI , and M asahiro N A GA SE
Department of Biology, F aculty of General Education, Gifu U niversity, Gifu, 501- 11, Japan
M any therm ophilic algae, principany blue-green algae and bacillariophyceae, can be found in the spas of Gifu prefed ure, as reported by Emoto in his study of the distribution of thermophilic organisms in spas throughout Japan (1). T hree bacteria have also been reported for Gifu prefecture : Gamom h μm 面 肖 α CH OL ODN Y , L ボ otM x tyickoga es CH OL ODN Y, and Lゆ tot加心 od mceα K UE TZI N G, all of which are rod shaped iron bacteria having filamentous structures. H owever, many bacteria are reported for other prefectures. T he discrepancy stimulated us to investigate the distribution of bacteria in Gifu prefecture and to characterize the biochemical behavior of thebacteria found. T heOkuhida spa area waschosen because it has the largest amount of hot w ater and the highest temperatures.
lnvestigation of therm ophiles is very interesting because it can yield information about thermophilic mechanisms. A lso, since the biologicaI substances produced by these organisms, such as enzymes, are very stable not only against heat but against other denaturing agents as we11(2ʼ3), research may lead to valuable applications of these substances to human life. ln fact, in Japan, 0 shima and lmahori(4) have characterized an eχtremely thermophilic bacterium isolated at M ine spa in lzu peninsu・ , Theym 鵬 theymo油 il鵬 strain H B8. T his bacterium has been em ployed to study the heat resisting mechanism of a cell membrane(5ʼ6), the properties ̀of cytochromer c oxidase(7), and a D N A binding protein(8). T his paper reports the results of a preliminary investigation into the grow th conditions and biochemical characteristics of yet- unisolated bacteria living in hot w ater in the Okuhida spa area。
M A TE R I A L S A N D M E TH OD S
Chemicals … … Polypeptone ( Daigoeiyo ChemicaI Co. Ltd., 0 saka) , yeast extrad ( Kyo- SU M M A R Y
T hirteen samples of hot w ater and one sample of hot sand were collected in the Okuhida spa area. T hirteen of them contained rod bacteria which grew in both a rich medium and a synthetic m edium . W ithout individual species or strains being isolated, the bacteria contained in each sample were analyzed for their grow th conditions and biochemical characteristics.
kuto Seiyaku C0. L td., T okyo) , casamino acids ( Difco L aboratories, Detroit, M ichigan, U . S.
A.) , agar (W akoPureChemical lndustries, Ltd., 0 saka) , andgelatin ( Hayashi PureChemical lndustries, Ltd., 0 saka) were obtained from commerciaI sources.
CoUed on oj̀ s叩 … … H ot water was c011ected at 13 sitesin theOkuhida spa area. T he tem perature of source w ater and the pH of the sam ples determ ined in the laboratory are show n in T able l. Portions ( about 100 ml) of sample w ater w ere immediately concentrated approχi- mately 10-times w ith a T oyo M embrane filter ( T ype T M - 4) . T he concentrated samples were frozen w ith dry ice and were lyophilized in the laboratory for preservation. 0 ne sample ( N 0.
14, T able l) of hot sand, whic!l was obtained at the bottom of a hot po01 ( N 0.7, T able l) , was treated in the same w ay except for concentration. 0 ne spoonfuI of w et sand w as subjected to lyophilization after being kept in pow dered dry ice. 々・
Culty e of badey仇 … … Five ml of the nutritional medium described by Oshima d ㎡(4).
wereadded tothelyophilized materia1s. Themedium contained 0.8% p01ypeptone, 0.4% yeast extract, and 0. 3% N aCI . T he pH of the mediUm w as adjusted at room temperature t0 7.5 with 1N s01utions of N aOH and H CI ( “PY N m edium ”) . Stainless-steel capped culture tubes containing the medium w ere stood in an oven kept at 60℃ . A fter one dayʼs incubation in the
ov en , 0 n e d r o p of th e tu r b id cu l tu r e w a s tr a n sf er r ed t o a f r esh m ed i u m . T h i s i n o cu l a ti o n w a s
repeated several times to remove the non-b1010gical turbidity which w as present in the sample water. Several one-ml portions of each culture were lyophilized and the ce11s were kept at - 50℃ over silica gel until use. W hen long term culturing ( several days or longer) was done, distilled w ater w as added to the .culture medium at appropriate intervals to compensate for w ater lost by evaporation. Cell grow th w as 血 onitored by m easuring absorbance at 650 nm in a spectrophotometer ( M ode1139, Hitachi Perkin-Elmer) using an F2 filter. A linear relation- ship betw een the cell concentration and the absorbancy w as observed in the range of absorban- ce between 0.02 and 0.12. By counting the cell number with a hematometer, it was estimated that 1.0 unit of absorbance corresponded t0 2.8 χ 108 ce11s per m1.
Micyoscol)ic obse刊at{om … … Flagella staining, spore staining, and gram staining were carried out by the m ethods of T oda, D orner, and H ucker, respectively. Ce11s w ere usually observed at a magnification of 600 0r 1,000。
R E SU L TS
Micyoscol)ic obseym tiom … … Only rod bacteria grew from any of thesampleseχcept from N 0. 10, the sample obtained from a w ater-pool directly connected to a w ell pump. A Il rods were around 0.5- 1.0 μm in diameter. The lengths of bacteria cultured in PY N medium are shown in T able l. T hese values are larger than those of aerobic mesophilic rods and similar to the values reported for an extrem ely therm ophilic bacterium (9). A ccurate com parison, however, will require more detailed investigiltion under strict conditions concerning such things as the composition of the culturemedium and age of cells observed. ,
Gyow tk of bade庖 … … Optimal conditions for the growth of the bacteria are also summarized in T able l, Detailed data coJlcem ing the growth-temperature relationship are shown in F ig. 1, in which the concentration of bacteria is eχpressed as absorbance at 650 nm . T he height of each curve attained in 27 hrsʼ stand culture roughly represents the growth of the
T able I Properties of sample w ater collected, grow th conditions, and length of rod bacteria
NN0591V.︻︼.()NN()冨な.︹一.()
Ce11 1ength (μm) Growth
Optium Temp( ℃) Source
Sample N 0.
Optium pH Temp( ℃) pH
8 。1 7. 9 7. 7 8 . 1 7. 1 7 . 5 7. 9 7. 9 7. 9 7. 6 7 . 7 7 . 9 8 . 4 ( 7. 9)
う05ぐ00000005056656655ra
1234〃a678910 ﹃り6792221596562153465476386567ぐ
6 . 0- 7 . 0 7 . 0 7. 0 7 . 0 7. 0 7 . 0- 7 . 5 7. 0 6. 5- 7 . 0 7. 5- 8 . 0
-
6 . 0, 9 . 0 7 . 0 7. 0 7. 0 11
Q乙34廿III
5 - 7 5 - 7 5 - 7 7 5 - 14 4 - 12 4 - 12 5 - 10 5 - 10
-
5 - 7 5 - 10 3 - 10 3 - 5
-
60 60 ( 50) 55 ( 50) 55
0. 1
0. 2
0. 1
0 0 0. 2 0. 4
■■㎜ ㎜㎜
T he pH of sample w ater w as determined at room temperature ( around 30℃ ) in the laboratory. T he values of the optim um pH for grow th are those adjusted at the room tem perature w hen the culture m edia were prepared.
Oj
翌()召にi.(一.()
0. 3
50
0 . 2
Fig. I Dependence of bacterial grow th on temperature.
Portions ( each 0.02- 0.10 ml) of a fully grown cultUre were inoculated into 5 ml of PY N medium and stood in w ater baths kept at the indicated tem perature. A fter 27 hrsʼ incubation, the absor- bance at 650 nm of a 3-times diluted culture w as determ ined and plotted against thetemperature. ln (A). 0 , sample N 0. 1 ; 0 , 2 ; ( ●, 3 ; ● , 4. 1n (B), 0 , 5 ; 0 , 6 ; (●, 7 ; ●, 8 ; △, 9, and in(C), 0 , 11 ; 0 , 12 に●, 13 ; ●, 14.
0. 1
TEMP ( ゜C) 0
0 . 3
55 60 65
TEMP ( ゜C)
70
0. 4
TEMP ( ゜C)
6 7 PH
5 8
Bacterial growth in the presence of 2% N aCl w as eχamined using PY N medium . Bacteria in all the sam ples eχcept N 0. 5, 6 and 12 could grow w ell at this concentration, but no bacteria
grew inanysamplewith5% NaCl inthePYN medium。
Biock micd ck mdeyistics of bαdey仇 … … T he gelatin liquefaction ability of the bacteria wastestedby inoculating onedrop of fully-growncultureof bacteriainto5 ml of PY N medium containing 3% gelatin. A fter 7 daysʼ incubation at 60℃ , the cultures w ere taken up into P asteur
pipets ( 7095B-9, Corning GlassW orks, New Y ork) andthepipetswerestood in ice-water for
20 m in. L iquefaction of the gelatin w as confirm ed by checking whether the culture dropped T able I I Summary of three biochemical characteristics eχamined
H 2S production
9 10
bacteria of each samp】e at the indjcated temperature, sincethegrowth of cells at each peak w 血s ascertained, by prolonged culturing, to be the maximum under the conditions. lt is clear that the bacteria contained in each sam ple grew best under tem perature conditions the sam e as or similar to those of the source w ater from which they came in all but a few cases ( N 0. 2, 6, and 9) . Bacteria in samples N 0. 3 and 12 presented two absorbance peaks, dearly indicating the
ptesence of at least tw o different species or strains of rod bacteria. G row th rates w ere also observed at various pH s. Bacteria in all the samples except for N 0. 11 showed nearly symm etric grow th curves ( data not shown) w ith one optim um pH value ( T able l ) . T w o optim um pH values w ere obtained for sam ple N 0 . 11, 0ne at pH 6.0 and the other at fairly alkaline pH 9.0 ( F ig. 2) .
0. 2
0 4
Fig. 2 D ependence on pH of grow th of bacteria contained in sample N 0. 11. Portions ( each 0.05 ml) of a fully grown culture of the bacteria in sample N 0. 11 w ere inoculated into 5 mls of PY N medium at varying pH s as indicated, and the cul・
ture tubes w ere stood at 60 ℃ . D eterm ination of absorbance w as carried out in the same way as in Fig. 1.
1
三︹︺召﹂4.[].()
+ + +
+
+ + + + + + + +
-
++++++++ 十一十
Generally ( 十 ) meanspositiveresult and ( - ) negativeresult. ln thesulfideproduction assay, a double or triple mark means stronger production of sulfide relative to that in single mark cases.
-
11 12 13 14 Sample N o.
+ + + + + K N 0 3
reduction
-
-
Gelatin liquefaction
++++++
-
+ +
+++
123456789
-
SI OW
-
fast fast fast
-
slow fast
-
sI ow
M annito1
-
fast fast
-
-
fast
-
-
sI ow
-
SI OW
-
S10W
-
SI OW SlOW SlOW
-
-
XVithOUt
down through the pipets or rem ained in them when the caps w ere removed. T he results are shown in T able I I .
N itrate-reducing ability of the bacteria w as investigated with 0. 1% K N 0 3 added to the medium . A fter 2 daysʼ culture of the bacteria at 60℃ , the presence of K N 0 2, the reduction product, was ascertained with α-naphthylamine and sulphanyl acid. Bacteria in 8 samples had reduced nitrate ( T able I I ) .
lndole production from polypeptone was tested after 2 daysʼ culture in PY N medium . T he presence of indole was detected by Ehrlichʼs method using p-dimethylaminobenzaldehyde and potassium peroxodisulfate. B acteria in only one sample, N 0. 14, had produced indole.
Urea decomposing ability w aseχamined with 2% urea added to the culturemedium . A fter the bacteria w ere well grown ( 2 daysʼ incubation) , 30 μl of 0.2% methylred solution w as added to the cultures. N o bacteria w ere observed to decom pose urea.
Production of H 2S w as eχamined with 1% L -cysteine-H Cl added to the culture medium . Strips of filter paper treated with a solution containing 10% lead acetateand 10% glycerol were hung in the culture tubes so that PbS w ould form on the strip once H 2S w as liberated from the cysteine by the bacteria and its black color indicate the occurrence of H 2S production. T hese results are aIso show n in T able I I .
U tilization of sugars by bacteria w as investigated by identifying acid production in the culture medium. T w ice diluted PY N medium , the pH of w hich w as adjusted to 7.0, w as supplemented with a sugar at a concentration of 0.5% and bromphenolblue as a pH indicator at a concentration of 0.002% . T he m edium ( 5 m l) w as inoculated w ith one drop of fresh culture of bacteria and incubated at 60℃ . Change in color to yellow w as observed daily, with the ユニ Ultssummarized in T ableIII . Thesameresultswereobtainedwhen asyntheticmedium for
yeast(lo) w as em ployed as a basal m edium w ithout either CaC12 0r glucose added, in place of the
T able I I I Summary of acid production assay
Sugars
M altose Sucrose
Glucose Sample
-
-
SlOW
9̀34111
fast
-
-
fast fast : color change in 2 days
slow : color change in 4 days
One drop of fresh culture of each sample w as inoculated into 5 ml of the test medium and the cells cultured at 60℃ .
-
-
fast fast
-
-
slow
一
一
SI OW S10W
-
-
S10W
-
fast
fast
n
fast fastPY N medium . A s seen in the table, bacteria of 5 samples ( N 0. 1, 5, 8, 12, and 13) produced no acid from either of the sugars tested.
A 11 the bacteria w ere aerobic and grew only on the surface of both liquid culture media and
agarmedia. Thustherewasnowaytotestwhetherthesebacteriaproducegasfromsugars。
DI SCU SSI ON
Only rod bacteria grew in a rich medium at 60℃ from w ater samples collected in the Okuhida spa area of Gifu prefecture. ln some samples, particularly in N os. 5- 7, filamentous structures w ere aIso observed. T his fact suggests that the iron bacteria reported by E m oto(1) arecontained in thesesamples. Biochemical characterization of thesebacteria wascarried out before any isolj tion of speciQs or strains in order to draw out all detectable and interesting characteristics of organisms. lt must be noted of course that this type of investigation may fail to uncover interesting characteristics of som e of the bacteria due to possible interference by the presence of other bacteria.
Results of experiments on growth conditions of the bacteria show ed that almost a11 0rganisms grew best under conditions the sam e as or very sim ilar to those of the source w ater in w hich they lived. T hus all bacteria observed in this study can be classifed as m oderate therm ophiles according to Oshim a(11). lt w as also shown that all bacteria prefer neutral pH except those in sam ple N 0 . 11. Casam ino acids added to the m edium at a concentration of
0. 6% in place of polypeptonewerealsogoodnutrientsfor thebacteriain all thesamplesshown
in T able l . B acteria in all the sam ples also grew w ell in a synthetic m edium (10) from w hich CaC12 was omitted so that pH could beadjusted to 7.0 without forming precipitates. lt was also confirmed using bacteria of sample N 0. 11 that vigorous shaking at 60℃ enhanced the 欧 owth of bacteria 4- 5 times.
T hat the temperatures of sample ( T able l) w ere low er than the values ( around 97℃ ) known for hot spring w ater in this area will have to be eχplained. Samples were collected in small, natural pools form ed where w ater spilled from springs or wells. Sample N 0. 10 had a high temperature, however, no organism grew from this sample under the conditionsemployed, since this sample w as obtained in an artificial pool built of concrete and connected to a hot w en pum p. N o b.acteria grew at tem peratures higier than 70℃ ( F ig. 1) and at tem peratures low er than 35℃ ( data not shown) .
lt isvery interesting that bacteria of sample N 0. 11 grew only with tw o optimum pH s. T his may simply imply that this sample contains two distinctly different bacteria. Even if so, why the tw o bacteria, having optimum pH for growth at 6.0 and 9.0, 0ccur in w ater of pH 7.7 w iII have to be considered. M oreover, that a therm ophile preferred an alkaline pH for growth is physiologically interesting and there is the pos951blility of utilizing the plToducts of these bacteria, since they seem to be stabl at an alkaline pH .
A gelatin liquefaction test showed positive results for alm ost all bacteria when gelatin w as added to the PY N medium at a concentration of 3% ( T able I I ) . Bacteria in only tw o samples
( N0. 1 and 11) grew in a PY N medium containing gelatin at a concentration of 30% andthese
also liquefied gelatin completely in 2- 3 days. T hese samples are likely to contain some interesting bacteria which produce proteases with high activities. ln fact, a culture m edium ofa bacterium isolated from sample N0. 11 was ascertained, after concentration, tobeeffective in hydrolyzing synthetic substrates for collagenase(12・13), so the bacterium may produce a collagenase. Purification and characterization of the hydrolyzing activity is now under w ay.
A lthough some problems are associated with the nitrate reductiori test employed here, it can safely besaid that bacteria showing positiveresults can reducenitratetonitrite. For the organisms w ith negative reSults, care m ust be taken : either they could not catalyze this step, or the nitrite formed did not accumulate in the culture medium at thetime of thetest, which probably would have been due to the activity of some of the components of the reduction
system . ʼ
T hese investigations into the growth properties and biochemical characteristics of therm o- philic bacteria have suggested at least the presence of m any species or strains of rods in hot w ater in the G ifu area, although only three have been reported(1). Secondly, som e of the organisms that will be isolated are likely to have utilizable products.
Ackllow legemeylts : T he authors are very grateful to Dr. N . N aiki for his useful advice throughout the investigation and to M s. M . L ynne R oecklein for her reading of the m anuscript。
R E FE R E N CE S
(1) Emoto, Y . ( 1965) List of bioIogical entities inhabiting thermal springs in Japan. IV. Thermal flora of Japan. J. Soc. Eng. Mineral Springs, Japan. 3 : 173- 182 ( in Japanese) .
(2) Fujita, S. C., 0 shima, T . and lmahori, K. ( 1976) Eur. J. Biochem. 64, 57- .
(3) Oshima, T ., Fujita, S. C., and lmahori, K. ( 1982) Methodsin Enzymology, Vol. 89, 335- 340.
(4) Oshima, T . and lmahori, K . ( 1974) lntem atl. J. System. Bacteriol. 24, 102- 112.
(5) W akayama, N . and Oshima,T . ( 1978) J. Biochem. 83, 1687- 1692.
(6) W akayama, N. ( 1978) J. Biochem. 83, 1693- 1686. j (7) Hon-nami, 1( . and Oshima, T . ( 1984) Biochemistry, 23, 454- 460.
(8) Zierer, R., Grote, M., Dijk, J., and W ilson,K. ( 1986) FEBS Lett. 194, 235- 241.
(9) Lechevalier, H. A. ( 1977) Hn dbook of Micyobiology, 2ndEdition ( Laskin, A. I . andLechevalier, H. A. eds) . CRC Press, lnc., pp. 237- 245.
帥 N aiki, N. and lwata, M . ( 1962) Sci.Rep. Fac. Liberal Arts and Educ. Gifu Univ. 3, 70- 75 (in Japanese) . (11) Oshima, T . ( 1978) “Thermophiles”, Tokyo Univ. Press, pp. 16- 36 ( in Japanese) .
(12) W Unsch, E. and Heidrich, H .-G. ( 1963) H oppe=Seylers Z. Phys101. Chem. 333, 149- 151. ʼ
㈲ Van W art, H. Eoand Steinbrink, D. R. ( 1981) Anal. Biochem. 113, 356- 365.
