Microbiology~oolo~
Published by Elsevier
FEMS
"/3 (19e~)
23-30
23
FEMSEC 00230
Oligotrophic properties of heterotrophic bacteria and in situ
heterotrophic activity in pelagic seawaters
Mitsuru Eguchi and Y u z a b u r o Ishida
Delmr:men: ~ Fisheries, Faculrjof Agriculture, Kyoro Unwersily, Kyott~d~p~n
Received5 October 1988
Revisionreceiv*d23 March 1989
ACcepted 7 April 1989
Key words: Oligotrophic bacteria: 14C-MPN method; Uptake kinetics; Glycine; Acetate
I. SUMMARY
The distribution of heterotrophic bacteria in
polluted coastal and unpolluted pelagic seawaters
was studied using a 14C-MPN method with either
five or seven kinds of taC-organie compounds as
substrates. The total number of heterotrophic
bacteria in pelagic waters ranged from 9.2 × 10 3
to 5.4 x 104 cells/m] and more than 85% of the
heterotrophic bacteria were represented by obligate ofigotrophs. In coastal waters, the number
of heterotrophs was one order of magnitude higher
(av. 3.5 × 10 s calls/nil), and eutrophic and facultatively oligotrophic bacteria were predominant.
Oligotrophs in pelagic waters had a high specificity for the utilization of amino acids, especially
glycine, and acetate-utilizing bacteria were scarce.
'The in situ maximum uptake rates of glutamate
and giycine were much higher than those of glycolate and acetate. Acetate uptake rates were extremely low or not detectable in pelagic waters.
The specificity of uptake kinetics is assumed to
Corres!mldemce to: M, Efucbi, Department of Fisheries, Fa¢ulvyof Agriculture, Kinki University,Nara 631. Japan.
depend on the existence of obligate oligotrophs
dominant bacteria in pelagic seawater.
2. I N T R O D U C T I O N
Recently. intprovements o! microscopical methods have clearly shown that most beterotrophic
bacteria are active in situ [1|. However, it is also
pointed out that large numbers of these active
bacteria can respire though not repwduce [2].
Therefore it is difficult to obtain physiological and
taxonomical information, e.g. about viability,
nutrient response and spexiation of the bacterial
assemblage with only microscopical methods. To
understand the physiological conditions and population changes of bacterial assemblages it is necessary to develop cultural methods. Ishida and
Kadota [3] reported on a new counting method
(14C-MPN method) to enumerate viable cells in
unpolluted oligotrophie waters. Using the modified t4C-MPN method, Ishida et at. [4] clarified
that obligately oligotrophic bacteria are the dominant population in the northern Lake Biwa. Furthermore, in the South China Sea and the West
Pacific Ocean, where the concentrations of utilizable organic substrates are lower than in the
0t68-6496/89/503.50 © 1989 Federation of European MicrobiologicalSocieties
24
northern Lake Biwa, the obligate oligotrophs
utilizing protein hydrolysat¢ or glucose have been
shown to predominate, while they could not utilize
acetate [5].
In this paper, we attempted to clarify the substrate specificity of oligotrophic bacteria by using
the 14C-MPN method with five or seven different
kinds of 14C-compounds. Further, we studied the
relationship between the existence of oligotroph
and heterotrophic activity involving five different
substrates in oligotrophic pelagic seawater (the
Kumano-nada; Owase Bay offshore), and in polluted coastal seawater (Owas¢ Bay).
3. MATERIALS AND METHODS
3.1. Sampfing locations
Seawater samples were collected during the following four cruises of the R / V Selsui-maru (Mie
University): 82-R-2 (April, 1982). 82-R-9 (September, 1982), 83-R-2 (May, 1983) and 84-R-2 (April,
14
/
14o"
x
~'E
.........
.
Ng. l. Samplinglocationsin Ow~seBay and 1heKumano-nada
Sea.
1984). The sampling locations (Sms.) are illustrated in Fig. 1. Subsurface (depth =0.5 m)
samples were collected with prccombustcd (450 °C
for 1 h) 1000-ml glass bottles. All samples were
stored on board of the ship at 5 ° C until bacteriological analysis. Inoculating procedures for the
enumeration of bacteria with the 14C-MPN
method and measurement of heterotrophic activity were initiated within 0.5 h after sampling.
3. 2. Determination of bacterial numbers
The numbers of viable heterotrophic bacteria in
seawater samples were determined by using the
14C-MPN method [4].
As described before [4,5]. two types of media,
InC-ST10-a and STI0 -I media, were used. The
14C-SIt0-4 medium contained 0.5 mg tryptieas¢
(BBL) and 0.05 rag yeast extract (Difco) per liter
of aged seawater (ASW). The concentrations of
organic nutrients in the ST10 -l medium were
1000 times higher than in the poor medium (]4CSTI0-4). Bacterial growth in ST10 -1 medium was
directly detected by turbidity and in 14C-ST10-4
medium growth was measured by the uptake of
~4C-compounds. 20 nCi of each 14C.organic compound, used in tracer amounts, was added to 5 ml
of the poor media (14C-ST10-4) which was then
autoclaved, The following ~4C-compounds were
added: L-[~4C(U)]-glutamate (285 mCi/mmol), D[~4C(U)]ghicose (268 mCi/mmol), [ ,4C(U)]acetatc
(59.6 mCi/mmo]), [~4C(U)]glycine (113 mCi/
mmol), L-[~4C(U)lserine (280 mCi/mmol), L-['C(U)]lcucin¢ (260 nlCi/mmol) and j~4C(U)]glycolate (8.8 mCi/mmol) in April 1982 and t4Cglutamate, ~4C-glucose, 14C-acetate, ~4C,glycine
and ~4C-glyoolalc during September 1982, April
1983 and April 1984,
After the incubation of inoculated ST10- i and
~4C-ST10-~ media at 2 0 ° C for 2 weeks and 4
weeks, resp~tively, the most probable number
(MPN) for five tubes was determined. Prior to the
determination of MPN By 14C-uptake, approx.
0.5-ml subsamples from the 4-week-old 14CST10-4dilution series were inoculated into freshly
prepared STI0-I medium (s(.~zond ST10 -L medium). The numbers of obligate oligotrophs,
faeultative ollgotrophs and eutrophs were calculated by the procedure described by lshida et aL
[4]. The populations of bacteria using °4C-STI0 4
medium (t4C-MPN) include both obligate and
facultativo oligotrophs, and while using ST10medium (T-MPN), the numbers of both facultative oligotrophs and ¢utrophs were counted.
Counts using the second STI0 t medium (2nd-TMPN) correspond to the number of facultative
oligotrophs. Obligate oligotrophs are obtained by
subtracting the 2ud-T-MPN from L4C-MPN, The
number of total beterotrophs is the sum of the
numbers of obligate oligotrophs, facultative
oligotrophs and eutrophs.
3,3. [n situ ]lererorrophic activity measuremem
The technique employed in this study was basically that of Parsons and Strickland [61, as modified by Wright and Hobble [7l. Two or five
different kinds of t4C-organic compounds were
used, In April 1982, 14C-glutamate and I'~C-acetate
were used, and 14C-glutamate, 14C-acetate, laCglucose, n4C-glycine and I'*C-glycolate were used
in September 1982 and April 1983. The specific
activities of the five kinds of IqC-:,ubstrates are
the same as mentioned above. The final concentration ranges for all substrates used in this
study are presented in Table i. Triplicate 5-ml
samples and one control were used at each of the
five substrate concentrations. Seawater samples
were incubated for 1 to 6 h at in situ tctaperatures
(_+I°C) in the dark. After incubation, treated
samples were filtered through G'.22-p,m pore size
membrane filters (Millipore corp.), and washed
three times with IO-ml portions of filtered seawater, The filters were dried and placed in scintillation vials containing 10 ml toluene fluor. Radioactivity was determined with a liquid scintillation
counter (Packard Tri-CARB Model 2425). The
Tabl01
Final concentrations used in the n~:asurcmcnt or the uptake
kinetics
Substrate
Concentration range(~M)
Acetate
0,08~t-0,420
Glutamate
0.057 0.285
Glucose
0.057-0.285
Glycolatg
0.277-1.385
(;lycine
0.067-0,335
V,,,, specific activity ( V ~ / c e l l ) was calculated
from total heterotrophic bacteria enumerated by
~4C-MPN method.
3. 4. Analysis of dissolved free amino acids in
setltfoler
150-m] samples of surface seawater of Sins. A
and D in April 1984 were filtered through precombusted (450°C for 1 h) Whatman G F / C glass
filters, and the filtrates were desalted and concemrated with a cation exchange resin (Dowex
50W-8X, H+). Individual dissolved free amino
acid concentrations in the treated samples were
determined with an automatic amino acid analyser
(HITACHI 835-50 Amino Acid Analyzer) [8].
Chemical oxygen demand (COD) of the
seawater samples was estimated from the amount
of KMnO. t consumed under alkaline ~eaction
(Japaneso Industrial Standard K 0101. 1979).
4. RESULTS
4.1. Distribution of oligotrophic bacteria
The bacterial numbers in surface waters at the
center {Sin, A) and the mouth (Sin. B) of Owasa
Bay, and offshore (Sins. C and D) on different
dates were counted using the MPN method with
I'~C-STI0-4 and STI0- I media (Fig. 2). The ratios
of obligate oligotrophs, facultative oligotrophs and
eutrophs to total heterotrophie bacteria are also
illustrated in Fig. 2. Sin. A was so polluted thai
the number of heterotrophic bacteria was always
more than 10 "~cells/mL and there was no significant difference between the 14C-MPN and the
T-MPN numbers. The C O D values of 1982 and
1983 samples were 1.56 and 2.68 02 mg/l. respcclively. In 1982 and 1984 samples, all the hereto,
trophic bacteria were represented by facultative
oligotrophs. The heterotrophs in 1983 samples at
Sm, A consisted of about 37% eatrophs, 26%
facultative oligotrophs and 37% obligate
oligotrophs. At Stn. B, where the polluted water of
Owase Bay was diluted with unpolluted water
from the Kumano-nada Sea, no appreciable difference between 14C-MPN and T-MPN was observed. About 13, 47 and 40% of heterotrophs
were represented as eutrophs, facultative
Water temp ('C)
'~ ~. -~
~.~..',.~, , ~ _ L ~ S
Table 2
l}actafial numbers (ml - l ) counted by using liquid media contalning organic nutrients in diff=cnt ~¢ntrations at :Sins, A
and D in April 1983
Date Stn.
Apr. 1982 A
Stn.
Media
STIO°
5T10- '
'4C-ST10-4
(approx.
3 8C/1)
(approx.
0.3 gC/l)
(approx.
0.~03 gC/I)
A
7.Sx104
5.4× 10s
5.4>:10 ~
D
4.9X 102
2.2X 103
5.4X 104
8
Apt. 1983 A
~
- ~
•
Apr. 19S/~ A
Ap~ lSOZ c
i
D
g
m
lil
m
5ep. 190Z O-I
O-2
I
Ape. 1983 C
Apr. 198L, 0
~
.
•
H
m
FI
n
I
•
•
most counts using these three m e d i a was S T I 0 °
<ST10-t=taC-ST10-4
at Sin. A, and S T I 0 °
< S T 1 0 - t < Z 4 C - S T 1 0 - 4 at Stn. D.
Fig. 3 shows the highest bacterial numbers and
the ratio of ofigotrophs utilizing each of the five or
*
Fig. 2, Comparison of the number of total h¢l¢rolrophs, the
ratio of 14C-MPN to T-MPN, and the ratio of obligate
oligotrophs, facubative oligotrophs and eutrophs to total hot*
erotrophs in Owasa Bay (Stng A and B) and the Kumano-nada
Sea (Sins. C and D). OO. obligate eligotraphs; FO, facultative
oligotrophs; E, cutrophs: WT, water ~r,perature; *. COD not
measurcu.
Date
~,t~
Sift
9
A
A~, 1983 A
oligotrophs and obligate oligotrophs, respectively,
O n the other hand, at Stns. C and D in the
Kumano-nada Sea, the numbers of heterotrophic
bacteria were I or 2 orders lower than those at
Sms. A and B, ranging from 9.2 × 103 to 5.4 × 104
c e l l s / m l (av, 2.2 × 104 cells/rid). The difference
between the t4C-MPN and T - M P N numbers was
so great that the ratio of obligate oligotrophs to
total heterotrophs was more than 8 5 ~ in all eases.
Eutrophic bacteria were not detected, The C O D
values were always less than 1 , 0 0 z m g / L In the
two water samples which were collected on the
starboard (Stn. D - l ) and the port (Stn, D-2) o f the
R / V Seisui-maru at the same time in September
1982, there were no distinct differences in the
number of heterotrophs or in the ratios of obligate
oligotrophs to total heterotrophs,
W e compared the bacterial counts using three
di[fercnt types of media (ST10°, ST10-t and t4CS T I 0 - 4 ) in April 1983 (Table 2). T h e o r d e r of
% Ot oligotrophs
~
~
~H.
,
Bacterial
numbe¢
~/m[)
..............
1'2x lOS
..............
1.1x 10s
' ............... 5"~ loS
tb
AIX, ~
C ~
........................~. . . . . . . . . . . . . . . . . .
S~,p,IS~,
o-1 , ~
I............... a2x10S
................
Ap~ l s ~
t,6x~:
,,.,o,
D
Qa©etl~ o 0tu~z.~; • Glutamate:
Fig, 3, Ratio of oli~trophi¢ bacteria utili~ng acetate, s l ~ e ,
glutamate, &iycolate,seriee, leuciae and 81ycine in Owas¢ Bay
(Stas, A and B) al~d the Kuroano-nada Sea (Sins, C and D),
Bacterial numbers ~re the highest cOUntSof each sample, and
stars indicate the counts that arc s~gniticandy lower ( p - 0.05)
than the highest counts.
~evcn t4C-organic compounds, in tracer amounts.
Distinct differences between the coastal (Sm. A)
and the pelagic waters (Stns. C and D) were
observed. At Sin. A, there was no statistically
significant differeno~ ( p > 0.05) among the numbers of bacteria utilizing any Of the substrates. But
at Stns. C and D, ther¢ were statistically significant differences ( p < 0 . 0 5 ) . The specificity for
substrates at Sin. B had both aspects of inland bay
(Sm. A) and pelagic waters (Sins. C and D).
A m o n g the 14C-compounds used in this study,
glycine always gave highest counts in April pelagic
water samples. Oligotrophs counted by using ~4C-
acetate were almost always significantly lower than
the highest counts in pelagic waters. T h i s tendency
may be related to in situ heterotrophie activity
and this relationship will be discussed later.
4.2. In situ heterotrophic activity
T h e heterolrophic uptake potential ( V ~ , ) , the
sum of the transport constant and the natural
substrate concentration ( K , + S n ) , the turnover
time (Tt) , and the V . ~ specific activity ( V ~ , / c e l l )
are shown in T a b l e 3. In general, Vn,~ was lower
and TI was longer in the samples with relatively
low numbers of heterotrophic bacteria (Sins. C
Table 3
Kinetic parameters for the uptake of acetate (OAt), glutamate (Olu;, glucose (GIc). glycolal¢ (GIyc) and glyeine (GIy) hy
heterotrophs in O.S-m deep wate~ of Owase [lay and the Kumano-nada Sea
Date
Sin.
April
0982)
A
B
C
D
September
(1982)
D-I
D-2
April
(1983)
A
D
Vmaa
(nmol/I/h)
Turnover
time (h)
K. + S.
(nmol/ll
V~/cell *
( × 10- s nmol/h/c¢ll)
64,2
15.3
38.4
3.5
679.g
S3.2
73"/.3
272.3
108.9
93.2
1011.6
515.1
2.5
3.6
2.2
20.5
9.4
~.8
34.8
187.9
33.8
213.t
t 550.8
5 233.7
217.1
46,2
193.7
4 979.0
262.0
1.5
0.8
7.7
9.2
0.S
367.5
1126.0
6369.6
264.2
86.8
843
3185.4
224.1
0.4
0.2
0.9
1.5
| .3
IS.5
3,2
78,9
4,2
69.2
0.3
I,,4.0
12.9
62,4
M.4
223.0
74.6
39.4
761.1
173.5
1134.5
1539.6
2':29.1
64.1
911.0
138.3
937.3
83.4
0.2
9.8
0.2
l.t
0.6
OAc
Glu
OAc
Glu
OAf
Glu
OAc
Gh
12.3
17.8
2.8
26,7
1.5
6.2
NMA
5.4
OAc
Glu
Gle
Gly¢
Gly
N MA
0.2
0,1
) .0
1.2
OAe
GIu
Olc
Glyc
Gly
NMA
0.2
0,1
OAc
GIu
GIc
Glyc
Gly
3,1
OAc
Glu
GIc
Gly¢
Gly
0.1
5.3
0.1
0,6
0.3
0.5
3.9
NMA, no measurable activity.
a /.z /celI were calculated from hetemtrophlc bacteria counted by the '4C-MPN method.
0.1
1.7
0.4
0.3
28
Table 4
The concentrations of dissolved free amino acids (nM) in
subsurface (depth m0.5 m) seawaters at Sins.A and D in April
19M
Glycine
Serine
Glutamicacid
Aspartic acid
Threonine
Alanine
Cystein
Vallne
Leucine
Isoleucine
Tyrosine
Phenylalanine
Omithine
Lysine
Histidin¢
Arsinine
Tnlal aminoacids
Stn. A
100.3
63.5
73.6
32.d
20.9
59.3
6.3
16.1
10.9
8.8
5.2
5.3
19.6
10.1
3.4
0.4
Sm.D
57.7
3a.5
19.4
14.8
6.2
19.2
2.0
4.4
5.2
1.6
4.1
3.3
11.4
4.7
2.8
0.0
436.2
191.1
and D, the Kumano-nada Sea) than in those with
higher numbers (Sms. A and B, Owase Bay). In
April 1982 and April 1983 samples, T~ values for
glutamate at Sms, C and D were 34.8-173.5 h laY.
97.2 h), and V.n~ values were 5.3-6.2 n m o l / 1 / h
lay. 5.6 nmol/l/h). These Tt values were about 10
times longer and Vma~ values were three times
lower than those at Sms. A and B. The difference
in acetate uptake was more remarkable. In April
1982 samples, Vmas values of acetate decreased
gradually from Sm. A to Sin, C, and eventually
the uptake of acetate at Stn. D was scarcely detected. Similar results were previously obtained in
the South China Sea, the West Pacific Ocean [5]
and the Antarctic Ocean ([9], Eguehi and Ishida,
in preparation), In September 1982, we collected
two seawater samples on the starboard (Sin. D-l)
and port (Stn. D-2) at Sin. D, The uptake of
acetate was also undetectahle in both of these
samples.
The concentrations of dissolved free amino
acids in seawater samples at Sins, A and D are
shown in Table 4, The total amounts of dissolved
free amino acids at Sin. A were more than two
times higher than at Sm. D. The concentrations of
giycine and glutamic acid were higher at Stn. A,
while giycine and serine were high in pelagic water
(Sm. D), as reported by Lee and Bada [10].
5. DISCUSSION
Our understanding of the rote of beterotrophic
bacteria in marine ecosystems has been improved
not only by the development of microscopical
methods to determine total bacterial number and
hiomass, but also by cultural methods to enumerate viable bacteria. The cultural methods give
valuable information about taxonomical and physiological aspects of bacteria, although the serious
disadvantage is that only a small fraction of the
total population is counted, especially by colony
counts on afar plates [11]. Among the many reasons for the differences between total and viable
counts, the concentration of nutrients is of particular importance. We have overcome some of the
difficulties described by Van Es and Meyer-Reil
[11] by using low-nutrient liquid media and testing
~4C-uptake from traces of labelled organic substrates added to the incubation tubes [4,5]. In this
reporl, qualitative and quantitative differences (cell
numbers, organic utilization and responses to
organic concentrations) among beterotrophic
bacteria between pelagic oligotrophic and coastal
eutrophic seawaters were clarified by using the
14C-MPN method and beterot~phic uptake kinetics measurement with several kinds of taC-compounds. In unpolluted pelagic seawater, such as at
Stns. C and D, the bacterial counts using the
14C-STI0-4 media (14C-MPN) were always one
or two orders higher than those using the conventional nutrient rich media (T-MPN) (Fig. 2, Table
2). The dominant population of viable bacteria in
the pelagic waters was oligotrophic, especially obligate oligotrophs which can be detected only by
using ultradilute media (e.g. 14C-ST10-4 medium),
Similar observations have been previously reported in the South China Sea and the West
Pacific Ocean [5].
The oligotrophic bacteria in pelagic waters preferred amino acids, especially glyeine, to acetate,
and the range of substrates that they can utilize is
much more narrow in comparison with hereto-
29
trophs in polluted coastal seawaters (Fig. 3). Such
a tendenc~J has also been observed in the Antarctic
Ocean (Eguchi and Ishida, in preparation). It has
also been reported that obligate oligotrophs isolated from Lake Biwa had a high specificity for
amino acids, such as glycine, glutamate and serine
[12]. This tendency was different from the characteristics of nutrient uptake for "modal oligoIrophs' proposed by Hirsch et al. [13]. The pelagic
environments where obligate ofigotrophs predominate are not only low in concentrations of
organic compounds, but are also small in variety
of them, as is shown in the dissolved free amino
acids observations (Table 4, [10]). Therefor',. it
may not always be necessary for oligotrophs to
have a broad range of subslrate uptake, It was
suggested that the predominance of glycine and
serine in seawaters (Table 4, [10]) depends on less
utilization by heterotrophs, because of a low energy yield per reel of these low molecular weight
amino acids [14]. However, our results showed
that giycine was a rather useful nutrient for heterotrophs in pelagic seawaters.
In this study, we used kinetic analysis based on
the Michaelis-Menten equation to determine the
rate of turvover of sabstrates in natural seawalers.
In pelagic waters, glutamate and glycine always
producec~ normal saturation curves and gave high
ttirnover rates, whereas the uptake rates of acctalc
were extr,'mely low. We assumed that this tendency in heterotrophic activity resulted from the
existence of obligate oligotrophs which have high
specificities for substrates (Fig. 3). It remains to
be proven whether the high specificity for substrates of pelagic oligotrophs is due to the inducibility of a large proportion of catabolic enzymes
[131 or not.
Wright [15] mentioned that the V~JAODC
(AODC: acridine orange direct count) is a good
measure of the average physiological state and
metabolic role of the bacteria. His conclusion
would support the hypothesis of several earlier
workers [16,17] that marine bacteria are adapted
to the conditions of nutrient starvation by becoming relatively inactive or dormant. AODC values
r~present total bacterial counts, and not viable
counts or active bacterial counts [1]. When the
specific activity (V,,~Jcell) is calculated, the major
problem is which bacterial counts are to be used
as the denominator [18]. if the Vm~/CeU were
calculated on the basis of heterotrophic bacteria
counted by the 14C-MPN method, except for
acetate, it could be conchidod that the potential
hetcrotsophic uptake activity per eel| in pelagic
waters is not at all inferior to that in coastal
waters (Table 3). This suggests the possibility that
naturally occurring oligottophs have the effective
uptake systems as "model oligotrophs" [13] have.
There may be two ideas concerning the living
forms of heterotrophie bacteria under ofigotrophlc
aquatic conditions. One is a notable conception,
that is, 'starvation-survivor [2]. The other is the
existence of oligotrophlc bacteria (e.g. see tees.
3-5, 13,19). These two ideas are not conflicting. In
ollgotrophlc waters, it is assumed that a majority
of eutrophs and a small part of the faeuhative
ollgotrophs are in the starvation-survlval stage,
non-growing, but sometimes not in an inactive
phase [20]. On the other hand, most obligate
oligotrophs and the rest of the facuhative
oligotrophs are probably growing actively in
oligotrophic waters.
Our observations show that bacterial populations physiologically change with the water bodies
of different trophic levels. However. what these
changes depend on (the physiological cell conditions, the taxonomical changes, or both) remains
to be determined. Martin and MacLeod [21] suggested that the existence of two broad classes such
as oligotrophs and eutrophs (or copiotrophs) differing intrinsically in their ability to grow at high
and low concentrations of nutrients was extremely
doubtful. They used 10 m g C / I as lower limit
concentration and detected bacterial growth by
turbidity. The organic concentration of our poor
media Q4C-STt0-4) is approx. 0.3 mgC/I, and at
this organic concentration bacterial rlnmbers do
not reach the l0 T cells/ml required for turbidity
measurement. It seems yet too early to decide
which of these two arguments is superior to the
other.
ACKNOWLEDGMENTS
We thank Drs. K. Hayashi and I. Sogawara of
Mie University for their kind help and advice, and
the captain, officers a n d crew of the R / V Seisuim a r u , Mie University, for their k i n d assistance in
s a m p l i n g d u r i n g the 82-R-9, 83-R-2 a n d 84-R-2
cruises. W e are also grateful to Drs. A. K a w a i a n d
H. K a d o t a of K i n k i University for helpful c o m ments, to D r . H . R a i of M a x - P l a n c k Ins t i t ut for
Lirrmologie, to D r . K . F u k a m i of K y o t o U n i v e r sity for critical r e a d i n g o f the m a n u s c r i p t , a n d t o
D r . S. H a r a of O s a k a U n i v e r s i t y for his k i n d help
with a m i n o acid analysis. T h i s s t u d y w a s p a r t l y
s u p p o r t e d b y f u n d s g r a n t e d b y the ~ i n i s t r y of
Education, J a p a n ( G r a n t N o . 57108016).
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