isolation of typical marine bacteria by dilution culture - growth, maintenance, and characteristics...

Vol. 59, No. 7APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1993, P. 2150-21600099-2240/93/072150-11$02.00/0Copyright ©) 1993, American Society for Microbiology

Isolation of Typical Marine Bacteria by Dilution Culture:Growth, Maintenance, and Characteristics of Isolates

under Laboratory ConditionsFRITS SCHUT,l* EGBERT J. DE VRIES,' JAN C. GOTITSCHAL,1 BETSY R. ROBERTSON,2

WIM HARDER,' RUDOLF A. PRINS,' AND DON K. BUTTON2Department ofMicrobiology, Biological Centre, University of Groningen, P.O. Box 14,

9751 NN Haren, The Netherlands, 1 and Institute ofMarine Science,University ofAlaska, Fairbanks, Alaska 99775 10802

Received 1 February 1993/Accepted 30 April 1993

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coastwere cultured in filtered autoclaved seawater following dilution to extinction. The populations present beforedilution varied from 0.11 x 109 to 1.07 x 109 cells per liter. The mean cell volume varied between 0.042 and0.074 ,um3, and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg ofDNA per cell. All threeparameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from veryhigh dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria.However, 15 of these isolates were eventually obtained from dilution cultures that could initially be culturedonly on very low-nutrient media and that could initially not form visible colonies on any of the agar mediatested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cellsin these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrientdeprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution offacultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half ofthe population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media,around 50%o could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilutioncultures obtained from samples diluted more than 2.5 x 105 times consisted of such obligate oligotrophs. Thesecultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 P,m3 andan apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria.Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agarplates were, with respect to cell volume and apparent DNA content, identical to the cells in the initiallyobligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these smallrod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids (a'A, 1,860 liters/g ofcells per h), but not for glucose or alanine as the sole source of carbon and energy (a'A, 200 liters/g of cellsper h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass inthe areas investigated.

Nondifferentiating heterotrophic marine bacteria are oftenthought to survive the oligotrophic conditions that prevail inoceans in a state of dormancy (78) or starvation (65, 74), i.e.,in a state of nongrowth. A possible rationale for this hypoth-esis is that although generally less than 1% of the cellspresent in natural seawater can be cultured and isolated onstandard laboratory media (13, 26, 48, 54), most of thebacterial population is metabolically active. The determina-tion of in situ rates of oxygen consumption (19, 21), aminoacid uptake (10, 20, 28, 49, 58), glucose uptake (6, 24),protein synthesis (53, 76), DNA synthesis (23, 27, 67), RNAsynthesis (50), and cell division (22, 36) and the determina-tion of electron transport system activity by using tetra-zolium salts (10, 72, 82) or fluorescein diacetate (60) and ofthe cell's energy charge (51), as well as the development ofactivity counts by microautoradiography (23, 43, 64, 79) ordirect viable counts by using the DNA gyrase inhibitornalidixic acid (54), have led to the conclusion that more than60% of the cells exhibit some sort of metabolic activity.However, the organisms responsible for these activities have

* Corresponding author.

yet to be identified and characterized, and as a result there islittle insight into the ecophysiological roles of individualsubpopulations. In this respect, community structure analy-sis of bacterioplankton populations using rRNA sequencesas a tool to identify different genotypes appears to be verypromising. It is now evident that numerous cells present inoceanic waters have 16S rRNA sequences not related to anyknown sequences from cultivated organisms (11, 30, 31, 75).The main problems in performing investigations into the

autecology and physiological properties of marine bacteriaare currently the isolation and cultivation of dominant ma-rine bacterial species. Whether the isolates obtained bycultural methods represent important indigenous bacterialspecies will remain unknown unless the culturability of thecells present in natural samples can be increased consider-ably or until it is proven that genetic and physiologicalcharacteristics for cultured isolates and indigenous cells aresimilar.As determined for natural assemblages of bacterioplank-

ton, the typical characteristics of marine bacteria includesmall cell size (9, 57, 76), even down to ultramicrobacterialsizes (cells with diameters less than 0.3 ,um or cell volumesless than 0.1 ,um3) (5, 61, 80), a low apparent DNA content

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ISOLATION OF OLIGOTROPHIC ULTRAMICROBAC7TERIA 2151

compared with that of laboratory-cultured cells (71), and ahigh uptake affinity for substrates (6, 15). Furthermore, thereare strong indications that marine bacteria require lowsubstrate concentrations for growth, i.e., that they areobligately oligotrophic. The definitions used throughout thisstudy are essentially the generally accepted ones (42), witholigotrophs defined as bacteria that have the ability to growat low substrate concentrations either obligately or faculta-tively and eutrophs defined as organisms restricted to growthat high substrate concentrations. Recently, the subject ofoligotrophy and ultramicrobacteria in relation to their sub-strate-sequestering abilities was discussed (15, 34). Gener-ally, platable cell counts (viable counts) from pelagic sam-ples increase significantly when a low-nutrient medium isused (2, 12, 17, 24, 45, 81). For this reason, the noncultura-bility of marine bacteria has been attributed to their obli-gately oligotrophic nature (24, 45), which would then implythat these bacteria are not dormant (78) but capable ofgrowth at ambient substrate concentrations (24, 46).So far, very few data have been collected on truly obli-

gately oligotrophic marine bacteria. In fact, it is not evenclear whether a demarcation line between oligotrophic andeutrophic bacteria exists at all. Eutrophic organisms can beretrained to become obligate oligotrophs (42), and obligateoligotrophs can be converted into facultative oligotrophs (61,63, 81).

Neither physiological traits nor morphological character-istics can be readily used to identify dominant marinebacteria. Normal isolates on plates have been shown to formviable but nonculturable cells (69, 73, 74 [and referencestherein]) as well as ultramicrocells (66, 77 [and referencestherein]) upon starvation or exposure to other stress condi-tions. In a study by Moyer and Morita (66), both the DNAcontent and cell size of a marine vibrio were reducedconsiderably upon prolonged nutrient deprivation, resultingin the morphology characteristic of indigenous pelagic bac-teria. However, since physiological properties of an isolateunder laboratory conditions will not necessarily reflect theorganism's natural state, information on the quantitativeimportance of the organism in its natural habitat and isola-tion in pure culture are needed before laboratory data can betaken back to the field.The aim of this study was to isolate dominant marine

bacteria by cultural methods. We used unamended filtered(0.2-,um-pore-size filter) and subsequently autoclaved sea-

water as dilution medium to study heterotrophic marinebacteria by applying dilutions to the extent of extinction. Atthe same time, we established a protocol for determining theviability of cells in the seawater sample. The ability togenerate cultures from very high (106-fold) seawater dilu-tions reflected the high level of viability of the cells presentin the inoculum. Viability values between 30 and 40% were

frequently observed. The theory, procedures, and initialresults of this promising technique have been describedelsewhere (16). In order to investigate the trophic characterof the cultures and isolates, we have determined the range ofcarbon source concentrations that can support the growth ofisolates by replica plating and by cultivation in syntheticseawater medium. Results indicate the presence of obli-gately and facultatively oligotrophic bacteria at the samplingtime and upon first cultivation. However, an unspecifiedadaptation leading to the ability to form colonies on solidmedium containing higher nutrient concentrations was ob-served for all obligately oligotrophic cultures tested. Iso-lates, once obtained, invariably represented facultatively

oligotrophic bacteria, i.e., bacteria able to grow at relativelyhigh and very low substrate concentrations.

Furthermore, we determined whether a representativedilution culture and an isolate obtained from such a cultureexhibited specific affinity values (15) comparable to thosegenerally observed for natural bacterioplankton (14, 34). It isconcluded that dilution cultures and isolates exhibit uptakeaffinities for mixed amino acids comparable to those ofnatural bacterioplankton.

MATERIALS AND METHODSSampling sites. Seawater samples were collected off the

Seward Marine Center pier at Resurrection Bay, Seward,Alaska, on 10 December 1989, 24 March 1990, and 28 August1990 (60°03'N, 149°25'W). The sampling depth was 10 m.

Experiments with the samples collected on these three datesare referred to as Resurrection Bay I, Resurrection Bay II,and Resurrection Bay III, respectively. North Sea water

samples were obtained at the oyster grounds approximately250 km north of the Dutch Island of Texel on 21 February1991, during a cruise of the MS Aurelia of The NetherlandsInstitute for Sea Research (54°75'N, 4°75'E). The samplingdepth was 20 m.

Preparation of dilution series and monitoring of growth.Dilution series were prepared and growth of the resultingdilution cultures was monitored as described previously (16).Unless stated otherwise, 20 tubes were inoculated for everydilution. For any tube, the observation frequency was lim-ited to a maximum of once every 3 to 4 days. This observa-tion frequency, together with the relatively high detectionlevel for populations in the dilution cultures (5 x 104cells perml), generally resulted in detection of cultures in the late logphase of growth at the earliest.

Subculturing and isolation. Subcultures of the dilutioncultures were made in filtered-autoclaved seawater (FAS)and in synthetic seawater medium (MPM), containing thefollowing (in grams per liter of Milli Q-purified water [Milli-pore Corp., Bedford, Mass.]): NaCl, 30.0; MgCl2- 6H20),1.0; Na2SO4, 4.0; KCl, 0.70; CaCl2. 2H2O, 0.15; NH4Cl,0.50; NaHCO3, 0.20; KBr, 0.10; SrCl2. 6H20, 0.04; H3B03,0.025; KF (Sigma Chemical Company, St. Louis, Mo.),0.001; morpholinepropanesulfonic acid (MOPS) buffer (Sig-ma) (pH 7.8), 2.09; KH2PO4, 0.27; trace element solution,1.0 ml/liter; vitamin solution, 2.0 ml/liter; and a carbonsource as mentioned in the text.The phosphates, trace elements, vitamins, and carbon

sources were added separately to the autoclaved basal salts.The trace element solution was developed from the data byGoldberg (32) and consisted of the following (in milligramsper liter of distilled water): Na2EDTA, 1,000; FeCl3 6H20,2,000; LiCl, 1,000; AlC13, 50; NaVO3. 14H20, 5; K2Cr207,0.15; MnCl2 4H20, 80; CoC12. 6H20, 5; NiCl2 6H20, 20;CuCl2, 20; ZnC12, 60; Na2SeO3 - 5H20, 15; RbCl, 150;Na2MoO4. 2H20, 75; SnC12 2H20, 1.5; KI, 80;BaC12. 2H20, 50; and Na2WO4 2H20, 15. The vitaminsolution was as previously described (39) and consisted ofthe following (in milligrams per liter of distilled water):para-aminobenzoic acid, 100; folic acid, 50; thioctic acid, 50;riboflavin, 100; thiamine, 200; nicotinic acid amide, 200;pyridoxamine, 500; panthothenic acid, 100; cobalamin, 100;and biotin, 20.

Dilution cultures were spread plated onto full-strengthmarine nutrient agar consisting of the following (in grams perliter of distilled water): nutrient broth (BBL MicrobiologySystems, Cockeysville, Md.), 8; NaCl, 27; and agar (BBL

VOL. 59, 1993

2152 SCHUT ET AL.

Microbiology Systems), 15. When growth occurred, isolateswere obtained from these plates. Dilution cultures that couldnot be cultured on marine nutrient agar or broth were

subcultured and maintained in FAS or MPM containing 0.5to 2.0 mg of Casamino Acids (Difco Laboratories, Detroit,Mich.) per liter. Spread plating of dilution cultures that couldnot be plated onto marine nutrient agar was also done on

ZoBell 2216E agar (MPM with 5 g of Bacto Peptone [Difco]per liter and 1 g of yeast extract [BBL] per liter) and on MPMagar without vitamins but with various concentrations of a

complex substrate mixture. This mixture was prepared as a

concentrated stock solution containing the following (perliter): D-glucose, 6.9 g; D-fructose, 6.9 g; D-galactose, 6.9 g;

Na-acetate, 9.5 g; L-lactic acid (90%), 6.4 ml (Fluka Chemie,Buchs, Switzerland); Na-glycolate, 11.3 g; Na-succinate, 8.1g (BDH Chemicals Ltd., Poole, England); mannitol, 7.0 g;

ethanol (96%), 7.0 ml; glycerol, 6.3 ml; Na-benzoate, 4.8 g;

salicylic acid, 4.6 g (BDH Chemicals Ltd.); Casamino Acids,76.5 g; and peptone, 29.7 g. The pH was set at 7.5 withNaOH. The carbon content of this stock was approximately100 g of carbon per liter. MPM agar plates were preparedwith various concentrations of the mixed-substrate stocksolution to yield organic carbon concentrations from 1 to10,000 mg of C per liter, called MPM1 to MPM10,000. Whenthese low-nutrient agar plates were used, the agar was

washed subsequently with ethanol (96%), acetone, and eth-anol (96%) and then washed three times with Milli Q-purifiedwater, when substantial amounts of sugars (1 mM reducingsugars in first H20 washing) and amino acids could beremoved from the agar.

Unless stated otherwise, all chemicals used were obtainedfrom Merck (Darmstadt, Germany) and were analyticalgrade. All vitamins were obtained from Sigma ChemicalCompany.Growth temperature of dilution cultures was 10°C in the

dark. Stationary-phase cultures were stored at 5°C in thedark. Spread plate incubations occurred in 10°C dark incu-bator rooms and at room temperature (20°C) in the light. Theisolates were then grown at 30°C in the dark.The population density and morphology of the cultures

were determined by flow cytometry and epifluorescencemicroscopy as described below.

Epifluorescence microscopy. For epifluorescence micros-copy, a modification of Porter and Feig's (70) nuclearstaining method was used. Subsamples (1 ml each) were

withdrawn aseptically from a culture, fixed in 0.2% (wt/vol)gluteraldehyde, and made permeable by treatment with 0.1%(vol/vol) Triton X-100 to improve stain penetration. Cellswere then stained for 15 min with 0.5 ,ug of 4',6-diamidino-2-phenylindole (DAPI) (Sigma Chemical Co.) per ml, col-lected onto a 0.1-,um-pore-size black, polycarbonate mem-

brane filter (Poretics Corp., Livermore, Calif.), and rinsedwith 4 ml of a prefiltered (0.2-,um-pore-size Nuclepore) 3%sodium chloride solution. The filter was fitted between a

microscope slide and a cover glass with immersion oil(Leitz). The cells were observed by first focusing on the filtersurface and slowly raising the focal point until the cells werein focus. Cells were counted at a magnification of x 1,000 byusing a Leitz dialux 20 EB microscope equipped with a

100-W Hg arc lamp and a 365-nm-wavelength optical band-pass filter. At least 10 fields were counted, or observationwas continued until 300 cells were counted.Flow cytometry. Flow cytometry was performed on a

Ortho Cytofluorograf IIS as described previously (71), ex-

cept that samples were preserved with 0.5% formaldehyde,stored cold, made permeable by treatment with 0.1% Triton

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Time (sec)FIG. 1. Time course of D-['4C]glucose uptake by strain RB2256

with (A) or without (-) 2-min preincubation with L-alanine (10 F.M)in order to energize the cells or with the protonophore CCCP (5 ,uM)(*). The glucose concentration was 20 nM. prot., protein.

X-100, and stained with DAPI (0.5 ,ug/ml) at 10°C for 1 hbefore analysis. Data were analyzed as described previously(16) and are plotted in bivariate histograms of apparent DNAper cell versus cell volume.

Uptake experiments and determination of specific affinity.Dilution cultures were subcultured in MPM on 2 mg ofCasamino Acids per liter. It was found that the low celldensities obtained by using these low substrate concentra-tions required rigorous pelleting procedures in order toobtain sufficient cell material. Therefore, cells were spun

down at 250,000 x g for 20 min, washed twice in MOPS-buffered MPM basal salts (pH 7.8) (MPMBS), resuspendedto a cell population of approximately 3.5 x 105 cells per ml(22 ,ug of cells per liter), and incubated with nanomolarconcentrations of 3H-labelled mixed amino acids (NET-250L-amino acid mixture, 28 Ci/mmol; NEN) at 10°C. Within 10min, 1-ml subsamples were withdrawn from the incubationculture and filtered through a 0.1-,um-pore-size Nucleporefilter. The filter was rinsed with 2 ml of MPM, dried, andcounted in a liquid scintillation counter, using a toluene-based scintillation cocktail.

Isolated strains were grown at 30°C in MPM with 2 mMD-glucose, 4 mM L-alanine, or 250 mg of Casamino Acids per

liter. Since cells tended to form little clumps at these highsubstrate concentrations and even formed strands on MPMplus glucose, sedimentation efficiencies were usually near

95% at 10,000 x g. However, by using electron microscopy,no significant differences were observed between the sizes ofindividual cells within such strands or clumps and those ofcells grown at low substrate concentrations (unpublishedresults). Cells were then harvested by centrifugation at

10,000 x g for 20 min at 4°C, washed in MPMBS (pH 7.8),and resuspended to a density of 3 mg of protein per ml. Cellswere stored on ice until measurement. For glucose uptake,10 pL1 of this cell suspension was resuspended in 100 p.l ofMPMBS (pH 7.8) and preincubated for 2 min at 30°C in thepresence of 10 p.M L-alanine in order to energize the cells.This procedure was necessary to facilitate immediate uptakeof the substrate tested (Fig. 1). Glucose was used to energizethe cells during alanine uptake. The uptake reaction was

Energ.

Not energ.

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ISOLATION OF OLIGOTROPHIC ULTRAMICROBACTERIA 2153

TABLE 1. Characteristics of bacterioplankton collected from two sites determined by flow cytometryz

Location Depth Date Population density MCV6 DNAC Carbon biomassd(m) (cells/ml, 106) (pm3) (fg/cell) (pg of C/liter)

Resurrection Bay 0 3 Apr. 89 0.71 0.042 4.7 10.4Resurrection Bay I 10 10 Dec. 89 0.83 0.048 2.5 13.9Resurrection Bay II 10 24 Mar. 90 0.20 0.052 2.7 3.6Resurrection Bay III 10 28 Aug. 90 1.07 0.074 3.7 27.7North Sea 10 16 Feb. 91 0.11 0.061 3.3 2.4

a The two sites were Resurrection Bay near Seward, Alaska, and near the oyster grounds, North Sea, The Netherlands.b MCV, mean cell volume.Apparent DNA content (population mean).

d The conversion factor 0.35 x 10-12 g of carbon per p.m3 (8) was used.

started with various concentrations of D-[U-'4C]glucose orL-[U-'4C]alanine (Amersham Corp., Amersham, UnitedKingdom). The reaction was stopped by quickly adding 3 mlof ice-cold MPMBS (pH 7.8). Cells were filtered through a0.2-,um-pore-size cellulose nitrate filter (BA 83; Schleicherand Schuell GmbH, Dassel, Germany) and rinsed with 3 mlof ice-cold MPMBS. Filters were immediately immersed inPackard Scintillator 299 scintillation cocktail and counted ina Packard 2000CA liquid scintillation counter.

Protein levels were determined by the method of Lowry etal. (59), using bovine serum albumin as the standard.

Specific affinity was calculated from the relationship a'A =Vma,IK,, where Vma. is the maximum specific uptake rateand K, is the half-saturation concentration for uptake afterconverting maximum uptake rates to units of substrateaccumulated per gram of cells (dry weight) per hour (15).

RESULTS

Enumeration of bacteria in raw seawater samples. Bacteri-oplankton populations in samples taken from ResurrectionBay ranged from a usual 0.20 x 106 to 1.07 x 10' cells per mlfor the Resurrection Bay experiments, as determined by flowcytometric analysis. The sampled North Sea population was0.11 x 10' cells per ml. On the basis of these data, bacterialbiomass was 7.0 to 82.3 p.g/liter (Table 1).Growth of cells in dilution culture. By epifluorescence

microscopy, the limit of detection of cells present in dilutionculture was determined to be approximately 5 x 104 cells perml. With final populations seldom exceeding 106/ml, growthwas generally not observed until the late log growth phase orafter 30 days (Fig. 2). Therefore, any morphological changein the appearance of the cells or change in populationcomposition during growth of the dilution culture could notbe detected. The following observations were all madeduring the Resurrection Bay experiments.

Several dominant cell types were observed in the differenttubes in which growth occurred. In various dilution cultures,a mixture of cell types was present. Besides very small andquite large rod-shaped organisms, spirilla, vibrios, and coccicould also be recognized. The cultures generated from thehighest dilution series consisted mostly of one single celltype, whereas those from the lower dilution series weregenerally mixed cultures with large cells (Table 2). Micro-scopic observations are supported by flow cytometric datashowing that cultures from lower dilutions also contain morecontour regions in the bivariate histogram (Table 2) (also seereference 16).The most frequently observed organism, in 9 of 17 positive

tubes of the highest dilution series (dilutions of 1 x 106 and5 x 105) during the Resurrection Bay II (March 1990)

experiment, was a small, straight rod, with a cell volume ofapproximately 0.05 to 0.06 ,um3 (length, 0.9 p.m; diameter,0.3 p.m), and an apparent DNA content of 1.0 to 1.5 fg ofDNA per cell (Fig. 3). It is likely that this organism wasovergrown in the lower dilution series by organisms that areable to attain higher maximum populations densities, sincemaximum population densities differed considerably be-tween cultures in which small rods were dominant andcultures in which vibrios, large rods, or a variety of differentcell types were present (Fig. 2). Furthermore, in cultures inwhich the larger rods were predominant, the presence ofsubpopulations consisting of smaller cells could often berecognized by flow cytometry (see Fig. 4 in reference 16).Some of these cells were small rods according to micro-scopic observations. Small rod-shaped organisms were thedominant cell type in over 50% of the cultures that weregenerated from the highest dilution series. Since all of thesecells were shown to have a uniform cell and colony morphol-ogy upon isolation (discussed below) and taking into accountthe fact that the overall viability of the sampled population

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Time (days)FIG. 2. Growth curves of representative examples of each of the

various cell types in dilution cultures obtained during the experi-ments with Resurrection Bay I (December 1989) and ResurrectionBay II (March 1990) samples. Cultures are identified according tothe dominant cell type present after 2 months at 10°C (mixed culture[0], vibrio culture [A], large-rod culture [E], and small-rod culture[0]). Small-rod cultures attain maximum population densities ofaround 0.25 x 106 cells per ml within the course of 2 months,whereas cultures with other dominant cell types attain maximumvalues of around 1.0 x 106 cells per ml. The horizontal line at log 4.5cells per ml represents the detection limit.

VOL. 59, 1993

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2154 SCHUT ET AL.

TABLE 2. Cell types in dilution cultures generated from the Resurrection Bay II (March 1990) experiment'

No. of cultures MCVb DNAC No. of subpopulationsdDilution Dominant cell type generated (xm3) (fg/cell) (histogram regions)

1 x 106 Small rods 2 0.051 1.0 1Large rods 1 0.231 2.2 3Cocci 1 0.054 2.2 1

S x 105 Small rods 7 0.058 1.4 1Large rods 3 0.153 2.5 3Cocci 1 NDe ND NDVibrios 2 0.096 3.4 2

2.5 x 105 Small rods 4 0.055 0.3 3Large rods 4 0.097 1.8 3Vibrios 3 0.148 2.7 2Coccoid (phage infectedf 2 ND ND ND

2.5 x 104 Vibrios 3 0.114 1.5 3Mixed 6 >0.2 >4 >4Coccoid (phage infectedf 1 ND ND ND

2.5 x 103 Mixed 10 >0.2 >4 >4

a Determined by flow cytometry and epifluorescence microscopy after 42 days of incubation at 10'C. For each dilution, a total of 20 tubes was inoculated. Whenthere was more than one culture of a cell type, MCV and DNA content data for only one culture are given.

b MCV, mean cell volume.c Apparent DNA content (population mean).d Number of subpopulations recognized by flow cytometry, specifically, the number of cell types present in one sample (see text; also see reference 16).e ND, not determined.f Possible phage infections leading to loss of the culture. The cell type was invariably coccoid.

was calculated to be between 29 and 69%, it was inferred culture and of isolated strains. (i) Resurrection Bay studies.that this small rod might form a very important subpopula- Extensive subculturing studies were performed on the dilu-tion within the natural bacterioplank-lton at the time of tion cultures of the Resurrection Bay II (March 1990) exper-sampling. iment. All dilution cultures venerated could be subculturedTrophic ranges of bacteria in raw seawater and in dilution in FAS, using inocula of 10 to 107 cells per tube. Subcul-

Apparent DNA content (fg/cell)

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Log DAPI-DNA fluorescence intensityFIG. 3. Bivariate apparent DNA content per cell versus cell volume histograms of a representative Resurrection Bay seawater sample

(December 1989) (A) and of a typical small-rod culture obtained by extinction dilution with an inoculum size of one cell per tube (B). Contourlines reflect population density. DNA content was measured as DAPI-stained DNA fluorescence and is therefore apparentDNA content (16).

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APPL. ENvIRON. MICROBIOL.

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TABLE 3. Cell populations present in dilution tubes of Resurrection Bay II (March 1990) and colony counts on various solid mediaa

Dilution culture Total Colony counts (CFU/ml of culture) on the following plate type:population MPM1 MPM10 MPM100 MPM1,000 MPM10,000 ZoBell 2216E

RB2256 6.2 x 105 7.9 x 103 2.2 x 10 1.2 x 104 0 0 6.7 x 105RB2259 4.8 x 105 1.0 x 105 1.6 x 105 9.3 x 104 1.1 x 105 0 5.0 x 105RB2510 1.0 x 106 1.6 x 104 4.8 x 105 4.7 x 105 2.1 x 105 0 4.5 x 105RB2512 1.1 x 106 2.9 x 105 5.4 x 105 4.2 x 105 3.5 x 105 0 1.0 X 106RB2518 3.6 x 105 6.2 x 103 1.3 x 105 9.2 x 103 0 0 8.4 x 105RB2519 8.0 x 105 3.2 x 104 4.1 x 105 2.8 x 105 3.2 x 105 0 4.7 x 105RB2109 1.1 x 106 7.2 x 104 5.0 x 105 1.5 x 105 5.0 x 103 0 9.6 x 105

a Dilution cultures stored at 5°C for 1 year were spread onto six agar plate types. Counts were obtained in duplicate, using six plate types as described inMaterials and Methods. Final CFU counts were obtained after 32 days of incubation at 20°C.

I Total counts (in cells per milliliter) present in dilution tubes immediately prior to plating. Counts were obtained by using epifluorescence microscopy asdescribed in Materials and Methods.

tures could even be obtained from the two highest dilutionseries after 2 years of storage at 5°C. All dilution culturesexcept those that contained small rod-shaped bacteria couldbe subcultured on full-strength marine nutrient agar or broth(eutrophic medium), and all dilution cultures could be cul-tured in synthetic seawater medium (MPM) containing 2 mgof Casamino Acids per liter (oligotrophic medium). There-fore, according to definitions used, only the small-rod-containing cultures harbored obligately oligotrophic bacteriaas the single cell type and all other cultures representedfacultative oligotrophs.Although these small-rod dilution cultures could not be

subcultured in high-nutrient medium, low-level (2-mg/liter)Casamino Acid additions allowed further growth of thepopulation. Progressive subculturing of these dilution cul-tures in MPM with 2 mg of Casamino Acids per liter yieldedstrands of organisms that often exceeded 100 pm in totallength. Transfer to FAS again yielded single cells.On repeated occasions dtiring and after growth of the

dilution culture, aliquots of the small-rod dilution cultureswere spread plated. Six months after the cultures hadreached the stationary growth phase, it was possible toobtain colonies from these cultures on ZoBell 2216E agarand on MPM agar. These colonies were barely visible to thenaked eye and could be properly observed and counted onlyby using a binocular at a magnification of x25. It tookapproximately 6 weeks at room temperature for these colo-nies to appear. This isolation procedure could be repeatedwith cultures that had been stored for 1 year at 5°C (Table 3).Colony counts differed with substrate concentration andtype of medium used. The highest numbers were obtained onZoBell 2216E agar, representing between 60 and 100% of thepopulation present in the dilution culture. Counts on thedefined complex substrate mixture in MPM agar were high-est with 10 mg of C per liter (MPM10). Colony countsrepresented around 50% of the population present in thedilution culture. No colony counts were obtained on MPMagar with 10,000 mg of C per liter (MPM10,000). The singlecolony type obtained from small-rod cultures of the Resur-rection Bay II experiment was a smooth, translucent, andyellow-pigmented colony. Flow cytometric analysis of purecultures generated from these colonies revealed that the cellvolume and apparent DNA content of these cells were

similar to those of the cells in the original dilution culture(Table 4). Incomplete segregation and strand formation werealso observed for these isolates during growth on 2 mMD-glucose, but not on 4 mM L-alanine.

(ii) North Sea studies. During the experiments with theNorth Sea samples, the trophic range of the organisms

present in the original seawater sample was investigated withvarious carbon concentrations in the dilution medium. Forthis purpose, FAS in the dilution tubes was supplementedwith the mixed-carbon-source stock solution also used forMPM agar to final dissolved organic carbon concentrationsof 5, 50, 500 and 5,000 mg of C per liter. After 2 months ofincubation at 10°C, of the 160 tubes containing FAS supple-mented with the four substrate concentrations and inocu-lated with 1 ml of seawater diluted 2 x 106 and 1 x 106 times,none produced cell populations above the detection level.This result indicated that the growth of dilution cultures wascompletely inhibited by the addition of 5 mg of C per liter.

Viable counts from the seawater sample on ZoBell 2216Eagar were 0.5 to 1% of DAPI-stained total counts (data notshown). Isolates obtained from direct spread plating of theNorth Sea sample on ZoBell 2216E agar were, however, ableto produce cultures in the carbon-supplemented dilutiontubes at all organic carbon concentrations tested, indicatingthat these organisms were not present in the inocula at thevery high dilutions used. These spread-plated isolates resem-bled the organisms generally obtained by standard isolationprocedures with a cell volume and an apparent DNA contentof 0.20 to 0.68 ,um3 and 3.8 to 4.6 fg per cell, respectively.From these observations, it was concluded that the contri-bution of cells present in the original North Sea sample andable to grow at high substrate concentrations (i.e., faculta-tive oligotrophs or eutrophs) to the total population was lessthan 1% and that the majority of the cells behaved as

obligately oligotrophic bacteria upon first cultivation in the

TABLE 4. Comparison of flow cytometry data of a raw seawatersample, a typical dilution culture, and an isolate of

Resurrection Bay II experiment*

Population density MCVb DNACMedium (cells/ml, 106) (Am3) (fg/cell)

Raw seawater sample 0.20 0.052 2.7FASd Dilution culture 0.71 0.072 1.1Subculture 0.24 0.051 1.0

MPMC1 mg of C/liter, Ala 7.2 0.06 1.7100 mg of C/liter, Ala 65.5 0.09 1.7

a Identification code of the dilution tube and isolate was RB2256. Thisorganism was one of the two small rod isolates from a dilution of 1 x 106(Table 2).

b Mean cell volume.c Apparent DNA content per cell (population mean).d Dilution culture in filtered autoclaved seawater.Isolate in synthetic seawater medium supplemented with alanine.

VOL. 59, 1993


dilution culture medium. The total culturable fraction of thisseawater sample was between 42 and 63% (16).

All 16 dilution cultures in unamended FAS generated fromthe North Sea sample could be subcultured in FAS, but noneof the dilution cultures could be subcultured in ZoBell 2216Ebroth. However, more than 1 year after reaching the station-ary growth phase, seven of these dilution cultures could beisolated on low-nutrient agars and ZoBell 2216E agar,whereas none of these cultures had produced colonies onany of the agars tested in the preceding period. The numbersof colonies appearing on spread plates accounted for approx-imately 80% of the cells present in the dilution cultures,according to direct microscopic counts (data not shown).This result showed that the remarkable adaptation periodeventually leading to the isolation of formerly obligatelyoligotrophic bacteria on plates that was observed during theResurrection Bay II experiment was reproducible. Onestrain (strain NS 1619) could be isolated 4 months afterreaching the stationary growth phase. This strain showedclose resemblance to the nine small, rod-shaped strains fromResurrection Bay II, both in cell and colony morphology,with a cell volume of 0.07 ,um3, an apparent DNA content of0.9 fg per cell, and yellow pigmentation. The strains thatwere isolated after 1 year were also rod shaped, but colonieswere not pigmented. Flow cytometric data of these cells arenot yet available.

All isolates eventually obtained from the North Sea exper-iment could be cultured on the total range of substrateconcentrations tested (FAS through ZoBell 2216E broth),and it was concluded that all isolates represented faculta-tively oligotrophic bacteria after isolation.

Specific affinity of dilution cultures and isolates. Uptakeexperiments were performed on a representative member ofthe small rod isolates of the Resurrection Bay II experiment,strain RB2256, and on a representative small-rod dilutionculture from this same experiment, culture RB2109.Time course uptake experiments with dilution cultures

revealed that the uptake of mixed amino acids was linear forup to 10 min. High cell density uptake experiments withstrain RB2256 were linear during the first 30 s (Fig. 1). Thepresence of 5 ,uM CCCP (carbonyl cyanide m-chlorophenyl-hydrazone) completely inhibited the uptake of glucose,indicating that uptake may be directly driven by protonmotive force. For chemostat-grown cells, preincubation withthe metabolizable substrate alanine was essential to allowimmediate uptake of glucose. This result may indicate thatcells became deenergized during the washing procedure thatpreceded the uptake experiment. Immediate uptake atequally high rates could also be accomplished by usingphenazine metasulfate and ascorbate as the energizing sys-tem. The increase in slope in the uptake curve by whole cellsthat were not preincubated with alanine implies gradual cellenergization by glucose metabolism (Fig. 1). It may thus beinferred that cell energization is necessary only in the courseof such short-term (1-min) uptake experiments but not in thelonger-term (10-min) uptake experiments with dilution cul-tures.For the isolated strain RB2256, half-saturation concentra-

tions for uptake (K,) and maximum specific uptake rates(Vma,) varied with the growth conditions. The lowest K,values for glucose were obtained at low dilution rates in thechemostat in the presence of a second substrate (alanine) (adetailed description of these experiments is being prepared).Eadie-Hofstee plots for glucose and alanine uptake duringnutrient-limited growth in continuous culture are presentedin Fig. 4.

50

,40

~30E 20 o

10

0 1 2 3 4

V/S (mL/min/mg prot.)FIG. 4. Eadie-Hofstee plots of substrate uptake data for glucose

(0) and alanine (-). Data were obtained with strain RB2256 grownon medium supplemented with glucose (2 mM) and alanine (4 mM)in continuous culture at a dilution rate of 0.025/h. prot., protein.

Calculated specific affinities for the dilution cultureRB2109 and for strain RB2256, together with data from theliterature for other marine bacteria are presented in Table 5.When bi- or multiphasic kinetics are described for purecultures, both high- and low-affinity systems are presented.

DISCUSSION

The occurrence of bacterial growth in unamended filtratesof seawater has been documented extensively. In order toobtain conditions as natural as possible, filtered seawater (4,35, 41, 47, 55, 57, 58, 76), autoclaved seawater (17), UV-irradiated seawater (37), and filtered-autoclaved seawater (7,38) have been used as the culture media for marine bacteria.In many studies, the mean cell size of the cultured bacteriais greater than that of cells normally observed in seawater (4,26, 52). This phenomenon may be the result of overgrowth ofthe dominant population by fast-growing atypical cells or theresult of a morphological alteration by the dominant cells.Conclusive evidence for either of these possibilities can beobtained only from genomic analysis of the cells.The small cell size and low apparent DNA content of the

small rod-shaped isolates obtained in the course of this study(even when grown in richer media) compared with the largecell size and high apparent DNA content of cells in culturesfrom lower dilution series suggest overgrowth by atypicalbacteria in low dilution series. Significant release of utiliz-able organic substrates may occur during filtration andsterilization of seawater (26, 33, 58). That this phenomenonoccurs is reflected in the fact that inocula of a few cells canreach populations of 106 cells per ml in unamended seawaterfrom which all cells have been removed by filtration and thathas subsequently been autoclaved (FAS medium). An eutro-phic organism, waiting for conditions to improve, may in thissystem overgrow an autochthonous oligotrophic bacteriumthat is not able to respond quickly (80) and has a lowmaximum specific growth rate. Extinction dilution is thenthe only remedy to obtain liquid cultures of dominantautochthonous organisms from seawater samples. Whethersubsequently obtained isolates truly represent dominantmarine bacteria remains to be determined by in situ oligo-nucleotide probe hybridization.

2156 SCHUT ET AL.


TABLE 5. Specific affinities for substrate uptakea by marine samples and known marine isolates calculated fromvarious references and of a dilution culture and isolate of the Resurrection Bay II experiment

V.. a' (liter/g of RerncSample or organism Region Substrate K, (nM) (nmol/min/ A ReferenceInstituteofOceanography, ~~~mg of cells) cells/h)

Seawater'-C Coastal seawater (Scripps Glucose 2.3d 0.23 6,000 6Institute of Oceanography,La Jolla, Calif.)

Kumano-nada Sea (Japan)

Aarhus Harbour (Denmark)

Seawaterlh,eGL-7f

RB2109 (dilution culture)

RB2256 (isolate)Vibric sp. strain S14

(starved)Serratia maninorubraAnt 300g

Vibric sp. strain S14 (logphase)

Vibrio sp. strain S14(starved)

Corynebacterium sp.strain 198

RB2256Pseudomonas sp. strain

486RB2256Vibrio sp. strain S14 (log

phase)Pseudomonas sp. strain

RP-303Alteromonas haloplanktis

(Pseudomonas sp.strain B16)

LN-155h

Ajstrup Beach (Denmark)

Sargasso SeaCoastal seawater (Scripps

Institute of Oceanography,La Jolla, Calif.)

Resurrection Bay (Seward,Alaska)

Resurrection BayBotany Bay (New SouthWales, Australia)

Pacific Ocean (off California)Antarctic convergence

Botany Bay

Botany Bay

Cook Inlet (Alaska)

Resurrection BayNansei Shoto area (Japan)

Resurrection BayBotany Bay

Nansei Shoto area (Japan)

Isolated from Pacific clams

Coastal seawater (ScrippsInstitute of Oceanogrphy,La Jolla, Calif.)

GlucoseGlucoseGlucoseGlucoseGlucoseGlucoseGlutamateGlycineGlutamateAlanineGlutamateAlanineAmino acidsGlucose

Amino acids

Amino acidsGlucose

GlucoseArginineArginineGlucose

LeucineLeucineGlucose

GlucoseGlucoseProlineAlanineLeucineLeucineGlucoseProlineAlanineAlanine

GlucoseGlucoseGlucose

32d150d

2,400d58,000d

590,000d85_194d46-911d83-262d671571831

75

200550

6,40017

4,5004,600

76020,0002,667

100-20,00013,600

2002,000-5,000760

20,0003,6001,800

46,000190,000

1806,600

180,000

0.68 1,2751.3 5204.2 10524 25110 110.7-2.9 494-9061.5-124 1,000-39,6002.2-34 1,600-7,7000.7 6471.3 4891.6 5,3002.1 4,100

6336.7 5,300

4,320

65.2

600.160.6637

1,860567

563550

9483

24

49

5838

This report

Preliminary data3

4029

3

5.6 442 626.7 20

388 14

4-206

0.75-17

1.710.70.151.36.9

20.8

0.010.050.39

92-22026

210133-180134322.5439

6.6

30.430.13

This report1

This report62

1

25

68

a Losses of label due to respiratory formation of CO2 are not taken into account.b Bacterial biomass is calculated by assuming commonly observed cell volumes of 0.065 ,um3, a volume-to-carbon (C) biomass conversion factor of 0.35 x 1012

g of C per pLm3 and assuming cell carbon to be 50% of cells (dry weight) (8).c Population not known, assumed density of 1 x 106 cells per ml.d Values are not corrected for the presence of ambient substrate and therefore reflect K, + S,.e Calculated from uptake rate of known population at added substrate concentration of 4.35 nM.f Biomass for GL-7 estimated at 0.9 x 10-14 g (dry weight) per cell (cell dimensions, 0.2 by 0.5 pLm. Formula for volume: rr2 (4/3r + L - W), where L is cell

length and W is cell width.g Biomass for Ant 300 estimated at 1.5 x 10-12 g (dry weight) per cell (cell volume, 2.2 p.m3 [29]).h Biomass for LN-155 estimated at 14 x 10-14 g (dry weight) per cell (cell volume, 0.20 p.m3 [18]).

An important part of the dilution cultures obtained con-tained bacteria with an obligately oligotrophic characterupon first subcultivation. After a period of up to 1 year in thestationary growth phase, during which the cultures were leftin the dark at 5°C, the ability to grow on medium with a

higher carbon content was displayed by 13 small-rod cul-tures from the Resurrection Bay II experiment and by 8small-rod cultures from the North Sea experiment. It is not

known whether specific mutations or other genetic changesinitiated this alteration in trophic character or whether(de)repression or induction mechanisms of metabolic or

energy-transducing pathways are at work here. Whateverthe case, the ability to adapt to eutrophic growth conditionsshows that the trophic character of seawater bacteria may besubject to alteration once they are separated from theirnatural environment, suggesting that obligate oligotrophy

Seawaterb

Seawatere

VOL. 59, 1993

(

APPL. ENvIRON. MICROBIOL.

reflects a life history, rather than a fixed physiologicalcharacteristic.

Since the extinction dilution method favors the growth ofpredominant species, the cultures generated from smallinocula (<10 cells) can be addressed as cultures of typicalmarine bacteria. The isolates obtained from these dilutionsare indeed similar in size and apparent DNA content to thoseof the bacteria normally present in seawater (Fig. 2 andTables 1 and 4). The 0.05 to 0.06 ,um3 cell volume is only 20to 25% that of a starved typical marine organism Pseudo-monas sp. strain T2 isolated on marine agar (71) and is equalto the remarkably low cell volume exhibited by Vibio sp.strain ANT 300 (volume of normal log-phase cell, 2.2 um3)during its starvation-survival response, imposed by a periodof nutrient-limited growth followed by a period of more than2 months of total nutrient deprivation (66). The apparentDNA content of 1.5 fg per cell is only 38% of a single-copyEschenichia coli genome (44). This fact, together with themean value of 2.7 fg per cell for the bacteria in the originalseawater sample (Table 1) shows the remarkably low appar-ent DNA contents of both indigenous marine bacteria andthe isolates described here.The absence of correlation between values for mean cell

volume and DNA content in raw seawater samples (Table 1)and in one of the isolated strains (Table 4) may be explainedby changes in population composition during the year ordifferences in the physiological state of the cells (also seereference 66). The low apparent DNA content of the smallrod isolates may, on the other hand, also reflect resistance toDAPI penetration by these cells or a systematic change inDNA fluorescence with cell size (16). Triton X-100 perme-abilization of cells improves DAPI fluorescence but is notalways consistent. Therefore, DNA content is reported as

apparent DNA content per cell. Hoechst 33258-based fluo-rescence measurements of cell extracts are planned. IfDNAmeasurements are indeed correct, these results indicate thatbacteria with the reported low cell volume and DNA contentare not necessarily exhibiting a starvation-survival response

(66) but may be actively growing bacteria.It is noteworthy that the small rod-shaped cells isolated in

the course of this study exhibit a high specific affinity formixed amino acids, while their affinity for glucose or alanine,supplied as the sole carbon and energy source does not setthem apart from other marine isolates. In fact, the affinitiesobtained for these two substrates would result in doublingtimes of up to half a year at concentrations in the environ-ment of around 10 nM (formula 23 in reference 14). At thismoment, specific affinity values for mixed-amino-acid up-

take by other described marine isolates are unknown, but thepossible presence of high values is of great interest andshould be investigated in the future. Multiple-substrate uti-lization would be the organism's only possibility to createfluxes of organic carbon that can support realistic growthrates. The reported specific affinity for mixed amino acids byRB2256 would enable this organism to multiply every 4 daysat ambient concentrations of 100 nM mixed amino acids. Inthis context, Law and Button (56) have shown the decreasein growth threshold concentrations for glucose in the chemo-stat when additional amino acids were supplied in themedium. They already suggested that specific affinities ofaround 400 liters/g of cells per hour for each component oftheir balanced mixture of 21 substrates would enable cells togrow with doubling times of a few days at oceanic substrateconcentrations.The remarkably high specific affinity value for glucose of

strain GL-7, as presented in Table 5, is somewhat doubtful.

The specific uptake rate by this organism was calculatedfrom reference 38, using the reported cell size of 0.2 by 0.5,um. However, cell size-to-biomass conversions are subjectto considerable error. A cell with essentially the samedimensions of 0.24 by 0.54 ,um or 0.16 by 0.46 ,um has a 53%larger or 40% lower cell volume, respectively. Such errorsmay result in a significant deviation in the calculated specificuptake rate. The same may be noted for Ant-300 and LN-155in Table 5.Although the values for specific affinities obtained from

pure cultures are generally somewhat lower than thosedetermined for natural samples, it is premature to concludeon the basis of these results that cells isolated thus far areessentially different from cells present in the environment.Physiological conditioning such as starvation of cells hasbeen shown to increase the substrate-sequestering abilitiesof Vibrio sp. strain S14 (Table 5). Growth conditions pre-vailing in the ocean will be very difficult to reproduce underlaboratory conditions, and data can never be realisticallyextrapolated to the oceanic situation when the mechanism ofobligate oligotrophy is not clearly understood. To achieveunderstanding, it is essential that representative organismsbe investigated, even if those organisms have lost theirobligately oligotrophic character. We are currently settingup for 16S rRNA sequence analysis, together with oligonu-cleotide probing of natural waters, to prove whether theisolates presented in this study are indeed an important partof the bacterioplankton population in the areas investigated.

ACKNOWLEDGMENTS

This work was supported in part by grants from the Foundationfor Sea Research (SOZ), the Netherlands, the Department ofBiology and the Bureau Buitenland of the University of Groningenin The Netherlands, the Royal Dutch Academy of Sciences, TheNetherlands Institute for Sea Research, the Biological Oceanogra-phy section of the U.S. National Science Foundation, and personalfunds of R. A. Prins.

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