Oligotrophic properties of heterotrophic bacteria and in situ heterotrophic activity in pelagic seawates

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 °
*

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

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

0.1 1.7

3.9 3,1

I,,4.0 12.9

62,4 M.4 223.0 74.6 39.4

0.1 5.3 0.1 0,6 0.3

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 OAc Glu GIc Gly¢ Gly

0.5

0.S

4,2 69.2

NMA, no measurable activity. a /.z /celI were calculated from hetemtrophlc bacteria counted by the '4C-MPN method.

0.3

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).

REFERENCES [1] Tabor, P.S. and Neihof, R.A. (1982) Improved microautcradiographic method to determine individual microorganisms active in substrate uptake in natural waters. Appl. Environ. Microbiol. 44, 945-953. [2] Mofita R.Y. (1982) Slarvatlon-survival of heterotrophs in the marine environment. Adv. Microb. Ecol. 6, 171-198. [3] Ishida, Y. and Kadola, H. (1979) A new method for enumeration of oligotrophic bacteria in lake water, Arch. Hydrobiol. Beih. 12, 77-85, [4] |shida, Y., Shlfiahara, K., Uchida, H. and Kadota, H. (1980) Distribution of obligately oligatrophic bacteria in Lake Biwa. Bull. Jpn. Soc. Sci. Fish. 46,1151-1158. iS] Ishida, y., Eguchi, M, and Kadota, H, (1986) E~gtenee o1 obligately oligotrophic bacteria as a dominant population in the South China Sea and the West Pacific Ocean. Mar. Ecol. Prog. Ser. 30, 197-203. [6} Parsons. T,R. and Strickland, J,D.H. (1962) On the pxoduction of partic-alate organic carbon by heterotrophic processes in seawater. Deep-Sea Pc.s, 8. 211-222, [7] Wright. R.T. and Hobble. J,E. (1966) Use of glucose and acetate by bacteria and algae in aquatic ecosystems, Ecol. ogy ';7,447-464. iS] AmonG. M.. Horn, S, and Toga, N. (1982) Utilization of dissolved amino acids in seawater by marine bacteria. Mar. Biol. 6S, 31-36,

[9] Oil[espie, P.A., Morita, R.Y. and Jones, L.P. (1976) The heterotrophic activity for amino acids, glucose and acetate in Antarctic waters. J. Ocear*ogr. Soc. Jpn. 32~ 74-82. [I0] Lee, C. and Soda, J.L. (1975) Amino acids in equatorial Pacific Ocean water. Earth Planet Sci. Left. 26, 61-68. [11] Van E.% F.B. and Meyer-RolL L.A. (1982) Biomass and metabolic activity of heterotrophic marine bacleria. Adv. Microb. Ecol. 6,111-170. [12] Ishida, Y. and Kadota, H. (1981) Growth patterns and substrate requirements of naturally occuriag obligate oligotrophs, Microb. Ecol. 7,123-130. [13] Hirsh, p. (1979) Life under conditions of low nutrient concentrations, in gtrategi~ of Microbial Life in Extreme Environments. Dahlem Konferenzen Life Sciences Research Report 13 (Shilo. M., Ed.) pp, 357-372. Voting Chemie, Weinhoim. 114] Andrews, p, and Williams, P,J,LeB, (1971) Hczerotrophlc utilization of dissolved organic compounds in the sea. III. Measurement of the oxidation rates and concentrations of glucose and amino acids in sea water. J. Mar. Biol. Ass. U,K. 51, 111-125. [15] Wright, R.T. (19781 Measurement and significance of specific activity in the heterotrophic bacteria of natural waters. AppL Environ. MicrobtoL 36, 297-305. [16} Novitsky. J.A. and Mofita. R,Y, (1977) Survival of a psychrophilic marine vibno under 10aS-term nutrient starvation. AppI. Environ. Microbiol. 33, 63S-641. [17] Stevenson, L.H. (1978) A case for bacterial dormancy in aquatic systems, Microb. Ecol. ld, 12/-133. [18] Simon, N. (1985) Specific uptake rates of amino acids by attached and free-living bacteria in a mesotrophic lake. AppL Environ, MicrobioL 49.1254-1259. [19] Poindexter, J,S. O981) Oligolrophy: fast and famine existence. Adv. Microb. Ecol. 5, 63-89. [20] Kjelleberg, S, Hermansson. M. and M,~rd~n, P, (1987) The transient pha~ between growth and no~rowlh of heterotrophi¢ bacteria, with emphasis on the marine eno vieonment. Ann. Roy, MicrobioL 41.25-49, [21] Martin. P. and MacLeod, R.A, (1984) Observations on the distinction between olisolrophic and eutrophic marine bacteria, Appl, Environ, MicrobioL 47, 1017-1022.