Vol. 58, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 359-3640099-2240/92/010359-06$02.00/0Copyright C) 1992, American Society for Microbiology

Bacterial Growth in the Cold: Evidence for an EnhancedSubstrate Requirement

W. J. WIEBE,l.2* W. M. SHELDON, JR.,2 3 AND L. R. POMEROY2'3Departments of Microbiology1 and Zoology3 and Institute of Ecology,2 University of Georgia, Athens, Georgia 30602

Received 8 August 1991/Accepted 14 October 1991

Growth responses and biovolume changes for four facultatively psychrophilic bacterial isolates fromConception Bay, Newfoundland, and the Arctic Ocean were examined at temperatures from -1.5 to 35°C, withsubstrate concentrations of 0.15, 1.5, and 1,500 mg of proteose peptone-yeast extract per liter. For twocultures, growth in 0.1, 1.0, and 1,000 mg of proline per liter was also examined. At 10 to 15°C and above,growth rates showed no marked effect of substrate concentration, while at -1.5 and 0°C, there was anincreasing requirement for organic nutrients, with generation times in low-nutrient media that were two tothree times longer than in high-nutrient media. Biovolume showed a clear dependence on substrateconcentration and quality; the largest cells were in the highest-nutrient media. Biovolume was also affected bytemperature; the largest cells were found at the lowest temperatures. These data have implications for bothfood web structure and carbon flow in cold waters and-for the effects of global climate change, since the changein growth rate is most dramatic at the lowest temperatures.

For over 100 years, it has been recognized that bacteriacan grow at temperatures near and below 0°C (8). Marinescientists, particularly, have been interested in the distribu-tion, physiology, and in situ activities of organisms growingat low temperatures, since more than 70% of the world'socean has a temperature of 50C or less (2). In recent years,microbial ecologists have investigated bacterial biomass,numbers, heterotrophic activities, and growth in a variety ofpolar environments, including sea ice, sediments, andcpastal and open ocean water."The results have been to some extent conflicting. Some

investigators report that bacteria in cold waters are asquantitatively important in marine food webs as they are intemperate seas (11, 15, 19), while others have found thatbacteria are often insignificant in processing organic matter(28, 32-34, 40). No doubt the location, time of year, andhabitat differences between studies account for much of thevariation. What seems clear, however, is that in a number ofinvestigations, bacteria, and thus the entire microbial foodweb, do not appear to be significant in carbon flux. Forexample, Smith et al. (40) calculated bacterial carbon pro-duction in sea ice algal blooms of 67.1 mg of C per m2 overthe entire period of the bloom, while net primary productionwas 1,942 mg of C per M2. Similarly, Pomeroy et al. (34)calculated that bacterial production accounted for 0.5% ofearly-bloom primary production and 5% of late-bloom pro-duction, while Nielsen and Richardson (28) in the North Seaand Schnack et al. (38) in the Southern Ocean reportedgreatly reduced microbial activity during algal blooms. Liand Dickie (25), while arguing that heterotrophic utilizationof products was not suppressed during the spring bloom inBedford Basin, Nova Scotia, showed clearly (their Fig. 1)that the lowest number and most uniform counts of bacteriaoccurred exactly at the time of highest chlorophyll a con-centration and lowest temperature.

It seemed paradoxical to us that bacterial abundance andproductivity should be low during algal blooms, based onobservations in temperate waters and on the abundant

* Corresponding author.

literature demonstrating that bacterial isolates are capable ofrapid growth at low temperatures. In reexamining the cultureliterature, one omission stood out, however: there werealmost no studies in which substrate quality or quantity wasinvestigated at low temperature and none we could find thatexamined bacterial growth and nutrient requirements below0°C. In virtually all experimental studies with cold-waterisolates, very high substrate concentrations (grams per liter)have been used in order to achieve turbidity or colonyformation. As discussed over a decade ago by Baross andMorita (2), the question is not whether bacteria can grow atlow temperatures given unlimited substrate, but ratherwhether they can grow at in situ substrate concentrations. Inthis study, we examined the growth and cell size of bacteriaisolated from waters of <00C over a range of substrateconcentrations and temperatures.

MATERIALS AND METHODS

Glassware. All glassware was acid washed (10% HCI) andfired at 450°C for 4 h.

Media. All media were made up in aged seawater; thisseawater alone did not support the growth of any isolate.The basic medium consisted of proteose peptone (PP) andyeast extract (YX), with the pH adjusted to 8.4. Concentra-tions of the test media were as follows: medium 1, 1 g of PPand 0.5 g of YX per liter; medium 2, 1 mg of PP and 0.5 mgof YX per liter; medium 3, 0.1 mg of PP and 0.05 mg of YXper liter. A proline medium was also used with 1 g, 1 mg, and0.1 mg/liter, pH 8.4, supplemented with 0.30 g of NaNO3,0.19 g of NH4C1, and 0.086 g of NaH2PO4 H2O per liter forthe 1.0-g proline medium and 10-3 and 10-4 these concen-trations for the 1.0-mg and 0.1-mg proline media, respec-tively.

All media were filter sterilized with seawater-rinsed Sar-torius 0.2-,um-pore-size filters. In a preliminary study, it wasfound that autoclaving significantly reduced the growth ratesand total cell yields of all bacteria at all substrate concentra-tions.

Cultures. All cultures were characterized to yield a pre-liminary identification. The criteria and tests are as follows:

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cell shape, motility, Gram stain, colony shape and color,acid and/or gas from glucose, catalase, and Kovac's oxidase.Methods are from Colwell and Wiebe (7). In addition, themodified Leifson 0-F test (39) was used. Stock cultures were

maintained in medium containing 1.5 mg of PP and YX perliter at 4°C and transferred weekly.

Cell counts and biovolume measurements. Bacteria were

fixed, stained with acridine orange (50 to 100 ppm, 3 min),and enumerated by direct counting with a Zeiss epifluores-cence microscope (10, 18). Nuclepore black polycarbonatefilters, 0.2-,um pore size, were used for all counting proce-

dures. The volumes filtered were adjusted to obtain at least10 cells per field; 25 to 50 fields were counted per filter. Cellsin mid-log phase were used to estimate biovolume duringsome growth trials. The length and width of at least 30 cellsper slide were measured with a calibrated ocular microme-ter. Cell volume was calculated by using the formula V =

(ii/4) x D2 X (L - D13), where V is volume, D is width, andL is length (24).

Experimental growth procedures. For every growth exper-

iment, the culture was first grown to mid- or late log phase inthe medium and at the temperature to be examined; inoculaused were in the mid- to late log phase. Duplicate flasks wereused, and each flask was inoculated with 0.5 x 103 to 1.0 X

103 bacteria per ml. Direct counts during growth were madeat least seven times before net growth ceased; samples were

immediately fixed with 2% filtered Formalin. Samples were

incubated in Neslab incubators, and flasks were manuallyagitated several times each day; temperatures were con-

trolled to within +0.05°C. Counting was done within 2 weeksof filtration. There was no loss of cells by this procedure ifthe filters were refrigerated (29).Growth rate determination. For each experiment, the

growth rate (,u) for cells in each flask was calculated bysimple linear regression of ln(number per milliliter) over on

time (hours) for five or six points in the linear portion of thegrowth curve. The growth rate and standard error were

determined from the slope parameter in the regressionanalysis. r2 was also calculated to measure the quality of thelinear fit. Generation times (gt) were calculated from thegrowth rates as follows: gt (h) = ln(2)/[L (h-').The data were then analyzed to determine the influence of

substrate concentration and temperature on growth rates bya two-way factorial analysis of variance (ANOVA) designwith replication and interaction. The data were log1o-trans-formed before performing the ANOVA, since sample vari-ances were not homogeneous and increased proportionatelywith the rates. This transformation normalized variances at

all temperature points and concentrations, satisfying theANOVA assumptions.

Pairwise comparisons between individual temperatures

and substrate levels were made with Tukey's W method (41).When there was a significant interaction between tempera-

ture and concentration factors, multiple comparisons were

preceded by analysis of simple effects (27). In this analysis,the effect of one factor is examined separately for each levelof the other factor, and the comparisonwise error rate iscontrolled by using terms from the overall ANOVA. This

procedure was used for most of the data, since the concen-

tration-temperature interaction was significant in nearly al!

experiments.

RESULTS

Organisms. N5 and N3 were isolated from 25-m depthduring the spring bloom in Conception Bay, Newfoundland,

in April 1988. 15 and 1120 were from surface water beneathsea ice at approximately 80°N latitude and 105°W longitude,in the Arctic Ocean. All four organisms had the followingcharacteristics: cell shape, curved rods; motile with polarflagella; Gram stain, negative; colony pigment, white; colonyshape, convex; produce acid but no gas in glucose medium;Kovac's oxidase positive; catalase positive; Leifson modi-fied 0-F test positive both aerobically and anaerobically at20 and 4°C. The preliminary assessment is that all fourisolates are in the genus Vibrio (43). All are classified aspsychrotrophs, following the definition of Baross and Morita(2).Growth rates. Bacterial growth rates may be examined in

a number of ways. Since our interest was to determinesubstrate effects over a range of temperatures, we haveplotted the data directly as generation time versus tempera-ture. As shown in Fig. 1, all the organisms, when grown onthe PP-YX media, had several features in common. First, at15 to 20°C, the growth rates are generally independent ofsubstrate concentration over a 104-fold range (1,500 mg to0.15 mg/liter). Second, while each organism displays slightlydifferent growth characteristics below 15°C, in every case, astemperature decreases, there is an increased substrate re-quirement for optimal growth; this effect is most dramaticbelow 4°C. In every case, temperature and substrate con-centration alone had a significant effect on growth rateoverall, and the interaction between substrate concentrationand temperature was significant for all cultures except I5.Two cultures, I120 and N5, were grown on proline as a

sole source of carbon (Fig. 2); generation times were approx-imately double those in comparable PP-YX media. How-ever, as shown in Fig. 2A and B, the trends are the same. At-1.5°C in the 0.1-mg proline medium, generation times were1.5 to 2 days, times that are similar to the calculated in situgrowth rates for bacteria in the Conception Bay algal bloomchlorophyll maximum (34). At 10°C, growth rate was inde-pendent of substrate concentration. ANOVA data show asimilar pattern for the PP-YX-grown cells (data not shown).

Cell size. Biovolumes were calculated for several cultures,as shown in Table 1. For N5 in PP media, cell volume waspositively correlated with substrate concentration at allincubation temperatures. Mean volumes of cells grown at0.15-, 1.5-, and 1,500-mg/liter concentrations were signifi-cantly different (P < 0.05, Scheffe's F test) due to increasesin both cell length and width with increasing concentration.Cell volumes generally increased with decreasing tempera-ture at all substrate concentrations. Cells grown at -1.5 and0°C were significantly larger than cells grown at 4, 10, or15°C. Changes in cell width but not length accounted for theincreases in cell volume at low temperature.N5 cells grown on 1,000 mg of proline per liter were

slightly larger than cells grown at the lower two concentra-tions at all incubation temperatures, but the differences wereonly statistically significant at -1.5°C. Mean volumes ofcells grown with 0.1 and 1.0 mg of proline per liter did notdiffer at any temperature. However, cell volume increaseddramatically with decreasing temperature at all substrateconcentrations, resulting in two- to threefold volume differ-ences between cells grown at -1.5 and 10°C. In contrast tothe results for PP-YX above, both cell length and widthincreased at low temperature.The influence of substrate concentration on cell volume

varied greatly, depending on the substrate used. Cells grownat the low and intermediate concentrations of both sub-strates were similar in size, while cells grown in 1,500-mgPP-YX medium were about 10 times larger than cells grown

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BACTERIAL GROWTH IN THE COLD 361

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Temperature, OCFIG. 1. Generation time versus temperature for cells grown in PP-YX media, as described in Materials and Methods. PP-YX amounts: +,

1,500 mg; A, 1.5 mg; O, 0.15 mg. (A) Isolate 1120 and (B) isolate 15 were from the Arctic Ocean; (C) isolate N5 and (D) isolate N3 were fromConception Bay, Newfoundland.

in the 1,000-mg proline medium, regardless of temperature.The temperature effect at a given substrate concentrationwas greater for proline-grown cells. For both substrates, theinfluence of temperature on cell volume was most pro-nounced at temperatures below 4°C.The other two isolates examined, 1120 and 15, showed

results similar to those for N5. Thus, cell size is mediumdependent as well as temperature dependent (Table 1).

DISCUSSIONThe taxonomy of cultivatable, heterotrophic, water col-

umn, marine bacteria from seasonally and permanently coldcoastal waters has been the subject of a number of investi-gations (e.g., reference 30 and references therein). A fewgenera dominate, and members of the genus Vibrio areperhaps the most commonly isolated organisms, as was thecase here. Of perhaps more interest and controversy iswhether obligate or temperature-tolerant psychrophiles pre-dominate and the role played by inclusion of different

organic compounds in the medium and their concentration.Recent articles by Noble et al. (30), Horowitz et al. (20),Ruger (36), and Upton and Nedwell (42) suggest that bacteriaisolated on low-nutrient media at low incubation tempera-tures are more nutritionally versatile, although the bacteriaisolated most often are not obligate psychrophiles, and theyoften grow on high-nutrient media. Carlucci et al. (4) alsonoted that most of their "low-nutrient" bacteria, isolated inaged seawater and washed agar, can grow under high-nutrient conditions.

Overall lack of predictable obligate psychrophily requiressome explanation, but none is available at present. Particu-larly troubling is the recognized low number of cultivatablebacteria compared with the total direct counts. Given therecent taxonomic study of Giovannoni et al. (13) and Brit-schgi and Giovannoni (3), the isolates described in theliterature (and here) may not be representative of marineenvironments, or it may be that under low-nutrient condi-tions, most individual cells become uncultivatable but re-

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main viable. It should be pointed out here, however, that theempirical results with natural, mixed populations of bacteriaalso show the same temperature-substrate relationship (34).Biovolume data showed clear dependence on substrate

concentration. Moreover, as shown for N5 and 1120, depen-dence was not just a concentration requirement but was alsorelated to the type of substrate (i.e., PP-YX versus proline,Table 1). Temperature also affected cell size, but generally ina more limited way. Volumes were smaller at high temper-

atures in both PP-YX and proline media. A similar result wasreported by Chrzanowski et al. (6) for both field measure-ments and with a Pseudomonas sp. in chemostat incuba-tions, using mineral salts plus 10 ig of yeast extract and 50,ug of glutamic acid per liter. They suggested that "a changein cell volume as a function of temperature is an intrinsicproperty of aquatic bacteria and is related to decreases ingeneration time." Their overall mean, average volume was0.153 p.m3, a value similar to that shown in Table 1 for all butthe 1,500-mg PP-YX medium. Palumbo et al. (31) examinedcell volumes of suspended bacteria in estuarine and coastalwaters; values ranged from 0.072 to 0.096 VLm3, again withinthe range reported here, except for the high-nutrient condi-tions. Interestingly, they found that cell volumes increasedwith increasing temperature and suggested that the largephytoplankton blooms associated with the temperature in-crease may have increased nutrient availability. Our datawould support this interpretation. Herbert and Bell (16)measured cell volumes of a marine psychrophile in chemo-stat-grown cells with glucose or lactose (1 and 0.5 g/liter,respectively) at 0, 8, and 15°C. Cell sizes in both mediadecreased with increasing temperature, and the investigatorsnoted that lactose-grown cells were in every case signifi-cantly smaller than those grown on glucose. Similarly,Herbert and Bhakoo (17) found that cell volume decreasedwith increasing temperature. In contrast, Frank et al. (9)grew several psychrophilic pseudomonads at 2 and 20°C innutrient broth and found no difference in cell size and severalother properties. It may be that the high nutrient concentra-tion that was used negated any temperature effect.One result from our study is unescapable: cell size is a

dynamic property of nutrient quality and concentration as

well as temperature and probably other factors as well. Inview of the importance of biovolume measurements inmarine ecological studies for calculating biomass (and car-bon) and as a factor in grazing selection (14), further inves-tigation of the factors that control cell volume are needed.There are numerous articles on the growth of bacteria at

low temperature or with low nutrient concentrations but fewarticles on the combined effects of substrate concentration,substrate type, and temperature on growth rates, particu-larly at low temperatures. Indeed, as Ingraham (21) pointedout in his review on the growth of Escherichia coli andSalmonella typhimurium, such studies are few, with noevidence that substrate concentration or type affects the

TABLE 1. Cell biovolumes

Addition Concn Mean cell biovolume (pLm3) + SEM at incubation temp:Organism to (gltr

medium (mg/liter) - 1.5C C 4C 10C 150C

N5 PP-YX 1,500 2.5 0.11 2.3 0.13 2.0 0.13 1.7 0.091 1.8 0.131.5 0.25 0.022 0.17 0.008 0.10 0.008 0.12 0.009 0.11 0.0080.15 0.14 0.011 0.11 + 0.008 0.073 0.006 0.070 0.005 0.068 0.008

L-Proline 1,000 0.26 0.018 0.20 0.014 0.094 0.007 0.088 0.006 NDa1.0 0.17 0.012 0.15 0.010 0.090 0.005 0.072 0.005 ND0.10 0.18 ± 0.012 0.18 0.011 0.076 0.005 0.064 0.004 ND

1120 PP-YX 1,500 2.3 ± 0.14 ND ND ND 1.0 ± 0.06

L-Proline 1,000 0.10 ± 0.006 0.086 ± 0.006 0.066 ± 0.006 0.077 ± 0.005 ND0.10 0.080 ± 0.005 0.081 ± 0.006 0.057 ± 0.004 0.045 ± 0.003 ND

I5 PP-YX 1,500 0.57 ± 0.037 ND 0.46 ± 0.035 ND 0.42 ± 0.026

0.15 0.11 ± 0.007 0.075 ± 0.005 0.071 ± 0.009 0.045 ± 0.006 0.077 ± 0.009

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BACTERIAL GROWTH IN THE COLD 363

lower growth temperature, but these factors can affect theupper temperature. The most complete discussion of thistopic for marine bacteria is by Herbert and Bell (16), whoexamined the growth characteristics of an obligately psy-chrophilic Vibrio sp. They found that sugars that supportedthe highest growth rates had the lowest K, values at 0°C,while carbohydrates that were less effective had the lowestKs values at 15°C. At 0°C, P'max varied from 0.01 to 0.022h-1, depending on the substrate, and in general increasedwith temperature, approximately doubling between 0 and15°C. They also calculated maintenance energy by usingglucose and found that it was 2% of the carbon input at 2°Cand 10% at 20°C. Similarly, Christian and Wiebe (5) notedthat growth efficiency decreased with increasing tempera-ture.

This study has implications for the extrapolation of labo-ratory studies of growth at low temperatures to field situa-tions. In almost all studies of psychrophiles and cold-tolerantbacteria, substrate concentrations have been in the grams-per-liter range, and most often the temperatures used havebeen from 2 to 5°C. Our results demonstrate that extrapola-tion of the results from former experiments to in situconditions at lower temperatures is at best problematic.Even the conditions in our experiments are less severe thanin the real world, since all of the substrate presented is labile,while most of the organic C in the ocean or lakes isrefractory (44). Our data demonstrate generation times forbacteria that are as high as or higher than those calculatedfor the Conception Bay spring blooms, using frequency ofdividing cells, thymidine incorporation into DNA, or leucineincorporation into protein (34).We have not identified the mechanism(s) responsible for

the temperature-substrate effect on growth rates. There havebeen suggestions that substrate transport across the cyto-plasmic membrane may be impaired at low temperature (37),that enzymes can be inactivated at temperatures near 0°C(35), and that synthesis of a number of proteins can beimpaired or blocked at low temperatures (1). In addition,ribosome function appears to be affected at low temperature(22). At present it is not possible to identify which of thesemechanisms is or are most likely. We have begun to inves-tigate these and other possibilities.One other factor that might affect the results reported here

is diffusion of substrate to cells. Diffusion changes withtemperature at about 2.5% per °C and is nearly linear (26).Thus, the rate is reduced by 75% over the temperature rangefrom -2 to 28°C. Generation times should approximatelydouble from this limiting factor. However, generation timeschanged by a factor of 10 to 20 over this temperature range,and thus diffusion alone could not account for our results.We are not, then, trying to extrapolate directly from our

culture data to environmental conditions; clearly, there arenumerous differences between culture conditions and thosein the sea. What we do believe, however, is that thesegrowth data support the view that at low temperatures,growth rate under low-nutrient conditions is much moredependent on substrate concentration than it is at highertemperatures. Although the dissolved substrates in seawaterare varied and to some degree undefined, the extant evi-dence suggests that labile substrates are usually nanomolarin total concentration (12, 23). Our results imply that growthof bacteria in most natural waters will be greatly reduced atlow temperature because of the quantity of substrates. Thesedata also have implication for global climate change, in thatthe change in growth rate is most dramatic at the lowesttemperatures. Thus, small increases in temperature would

be predicted to have a disproportionate effect on in situbacterial growth in cold waters.

ACKNOWLEDGMENT

This work was supported by U.S. National Science Foundationgrant OCE 8709809 to L.R.P. and W.J.W.

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