Vol. 36, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1978, P. 297-3050099-2240/78/0036-0297$02.00Copyright © 1978 American Society for Microbiology

Measurement and Significance of Specific Activity in theHeterotrophic Bacteria of Natural Waters

RICHARD T. WRIGHT

Department of Biology, Gordon College, Wenham, Massachusetts 01984

Received for publication 15 February 1978

It is now possible to obtain accurate total counts of the bacteria of naturalwaters with the use of acridine orange staining and epifluorescence microscopy.This approach can be coupled to highly sensitive measurements of heterotrophicactivity using radioisotopes. To accomplish this, three variations of a "specificactivity index" are suggested, based on different approaches to measuring heter-otrophic activity with radiolabeled organic solutes. The denominator of eachindex is the direct count of bacteria from a given natural sample. Three numer-

ators are presented, each of which has been shown to vary directly with hetero-trophic bacterial activity: Vm.x, turnover rate, and direct uptake (at high substrateconcentrations). Each approach is illustrated with data from estuarine and coastalwaters of northeastern Massachusetts. The data show major differences in specificactivity that accompany such habitat differences as distances within or offshorefrom an estuary and vertical location in the water column. These and other datasuggest that specific activity is a valid indicator of the physiological state andmetabolic role of the bacteria. Some evidence is presented in support of thehypothesis that the natural bacteria are adapted to conditions of nutrient star-vation by becoming "dormant," existing for an unknown period of time in a

reversible physiological state that reflects the availability of organic nutrients.

Bacteria in natural waters and sediments havelong been considered important agents of or-

ganic matter decomposition and mineral regen-eration, as well as food for higher organisms.Until fairly recently, however, information on

their activities and numbers was severely limitedby the available methodology. Numbers were

measured by "viable counts" on agar media, andactivity measurements consisted of laboratorytests of the ability of cultured forms to performvarious biochemical or mineral transformations.Recent advances in fluorescence microscopy,staining, and filter technology have led to theacridine orange direct count (AODC), using Nu-clepore filters, as described most recently byHobbie et al. (13). For measuring the activity ofthe natural heterotrophic bacteria, methodsbased on the uptake of 4C- and 3H-labeled or-

ganic solutes have proven adequate even in themost oligotrophic waters (see 28 for a summary

of these methods). The result is that there is

now available both a sensitive measure of natu-ral heterotrophic activity and an accuratemethod for counting the bacteria. Combiningthese two approaches is an obvious step, onethat may yield new insights into the functioningof natural bacteria.

Several workers have already used this basic

idea in their studies of heterotrophic activity innatural waters. Azam and Hodson (1) relatedglucose uptake to ATP-determined biomass inorder to evaluate the relative uptake of a givensize fraction as separated by Nuclepore filters.Their "relative metabolic activity" is dimension-less and compares the activity of a fraction tototal activity within a natural water sample; also,biomass is calculated by ATP measurements,which are not specific for bacteria. The mainthrust of this paper was to show that over 90%of heterotrophic activity in a variety of marinewaters was by organisms passing through a 1-pmfilter, obviously bacteria.Fry and Ramsey (9) used an activity index

based on uptake kinetics, V,,, per bacterium, intheir work on the heterotrophic activity of fresh-water epiphytic bacteria. Their index is given inmicrograms per liter per hour x 107 and appearsto be in error because of the inclusion of a

volume dimension. It is also several orders ofmagnitude too high. In this work autoradiogra-phy was used to determine the proportion oftotal bacteria actually metabolizing the sub-strate used. This is also a valuable approach toheterotrophic activity, one that has been usedby several workers to show that a variable pro-

portion of the total number of bacteria (deter-297

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mined by direct count) is metabolizing a givensubstrate (7, 14, 22). It is a qualitative approach,however, and has many technical problems thatrelate to concentrations of substrate, time ofexposure to film, and the counting of radio-graphic spots on filters.The present paper speaks to the need for a

quantitative expression of the relationship be-tween heterotrophic activity and the bacteriaresponsible for that activity. I will present threevariations of a "specific activity index," based ondifferent approaches to measuring heterotrophicactivity. I will then show some results whichindicate the usefulness of this combined ap-proach.

THEORETICAL CONSIDERATIONSThe concept of specific activity is used in

enzymology to relate the amount or rate of en-zyme activity to the more or less pure proteincontaining the enzyme. It seems appropriate toadopt this concept for relating heterotrophicactivity to the organisms responsible for thatactivity (I am grateful to D. W. Schindler forthis suggestion). Throughout the paper, the termspecific activity index represents the use of theconcept of specific activity in a quantitative re-

lationship.The bacteria can be represented in the rela-

tionship in several ways: numbers, volume, andbiomass. Two approaches are available for meas-uring numbers: the direct count and the platecount on some selected medium. Plate countsoccasionally correlate with heterotrophic activ-ity (23), but it is well known that the plate countyields only a small fraction of the total numberof bacteria present in a sample (15). There iscertainly a use for plate counts, but the evidenceclearly shows that the plate count does notmeasure either total numbers or living, activebacteria (6, 17). The direct count, on the otherhand, is a rapid and simple method that yieldsa count of all bacteria present; it does not distin-guish among living, dormant, and dead bacteria,and it does not readily yield information on thedifferent sizes of the bacteria in a sample. How-ever, in the opinion of this worker, the directcount using acridine orange and epifluorescenceis much less equivocal than the plate count andwill ultimately yield more information on theheterotrophic bacteria as it is more widely used.Volume measurements are tedious and impre-

cise with the light microscope and more precisebut also tedious and costly with the scanningelectron microscope (4). Biomass estimationscan be made from volume measurements, or

from a new method using Limulus amebocytelysate to measure bacterial lipopolysaccharide

(25). This latter method is very promising buthas the disadvantages of being costly and as yetnot widely tested. The advantages seem to bewith the direct count (AODC), which correlateswell with the Limulus amebocyte lysate method.The counts from natural waters range from 108to 1010 liter-' (5, 10; unpublished data); hence, aunit of 109 bacteria liter-' would appear to bethe most useful for the denominator of the spe-cific activity index.The numerator of the index should be some

measure that varies directly with heterotrophicbacterial activity and is sensitive to activity atnatural substrate concentrations. Three indexesare proposed, based on different approaches tothe application of radiolabeled organic solutesto natural waters (28).

(i) V,.-kinetic approach. The kinetic ap-proach to measuring heterotrophic uptake oforganic solutes uses a series of concentrations ofthe labeled solute in the microgram-per-literrange (29, 30). This approach generates Vm,, aninterpolated parameter that implies a full-capac-ity uptake rate for the substrate and populationin question. If respired '4CO2 is measured, themethod gives Vmax for total uptake of a solute. Ifnot, Vm,. for assimilation is obtained. Values forVm. range considerably, from 10-4 to 101 ,ugliter-' h-'. For a Vm,,x specific activity index, auseful set of units would be 10-3 ,ug liter-' h-1,yielding the index: Vmax (10-3 ,ug liter-'h-')/AODC (109 cells liter-'), or 10-12 ,ug h-cell'.

(ii) Turnover rate-tracer approach. Iflabeled substrate is used at the tracer level, it ispossible to obtain the natural turnover time fora substrate with a measurement at only oneconcentration (2, 26). The kinetic approach alsoyields the natural turnover time but requiresmore samples and is of limited use in oligo-trophic waters. The tracer approach involvesadding 3H- or '4C-labeled substrate at low con-centrations (<1 ,tg of substrate liter-') and thenmeasuring the fraction of added substrate thatwas assimilated or respired or both. It should beemphasized that this approach is valid only ifadded substrate is substantially lower in concen-tration than naturally occurring substrate.Gocke (10) has compared the turnover timesobtained via the kinetic approach with thosedetermined with a single low concentration. Theresults show good agreement between the twoapproaches in all but the most oligotrophic ma-rine waters, the advantages of one or the otherdepending on a tradeoff of simplicity for thetracer approach versus the additional data pro-vided by the kinetic approach, namely, Vm. and(K, + S.) (28).Natural turnover time is defined as the num-

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ber of hours required for the existing hetero-trophic population to take up and/or respire aquantity of substrate equal in concentration tothe existing concentration. The more active thepopulation, therefore, the shorter the naturalturnover time will be; it is an inverse function ofheterotrophic uptake and as such would be awk-ward to use in an activity index. The simplestdevice is to use the inverse of turnover time.This parameter has been called "relative uptakerate" (2) or "respiration rate" when only respired"'CO2 was measured (26) and has units of h-'. Itis a measure of the percentage of available sub-strate taken up per unit of time. Odum (21) callsthis measurement "turnover rate," a term thatseems to be less confusing and therefore prefer-able. Since turnover is a function of uptake andsubstrate concentration, variations in both ofthese parameters will influence the measuredturnover rate. However, uptake will vary overseveral orders of magnitude, quite comparableto the variation in Vma, values, whereas sub-strate concentrations in natural waters typicallyshow little or no variation (5, 31). Turnover ratesrange from high values of 1 h-' or more in highlyeutrophic, warm waters to l0-5 h-' in extremelyoligotrophic waters like the open oceans. For usein a specific activity index, a turnover rate of 103h-1 is appropriate, giving an index: turnover rate(103 h-')/AODC (109 cells liter-) or 10-6 h-cell-' liters.

(iii) Direct uptake measurement. If thenatural substrate concentration is measured in-dependently, a direct calculation of v., the nat-ural uptake velocity, can be made with eitherthe kinetic approach or the tracer approach (28).This is probably the best heterotrophic activitymeasurement, but it is also the most difficult toobtain. If it were used in a specific activity index,the units would be the same as for the Vmaxindex calculations.However, if substrate is added in concentra-

tions above 100 ,ug liter-', it is legitimate toignore the unknown concentration of naturallyoccurring substrate in calculating subsequentuptake (28). This approach does not measurenatural activity in the sense of in situ rates orsubstrate turnover because of the unnaturallyhigh substrate concentration. Because this ap-proach uses a single concentration and directcalculation, it is not concerned with the kineticsof uptake. It might be used, for example, intesting the response of a natural population toorganic enrichment over a period of days (3).The heterotrophic activity is then expressed asa rate v = [f (A)]/t, where v = uptake and/orrespiration of the substrate, f = the fraction ofavailable labeled substrate taken up, A is theadded substrate concentration, and t is the in-

cubation time. Note that natural substrate con-centration, S,,, is ignored in the calculation. Incombination with bacteria numbers, this specificactivity index would have the same units as theVmax index: 10-12 ,ug h-' cell-'.One major factor in measuring and comparing

values for heterotrophic activity is the influenceof temperature on the measured values. In avery extensive study of the temperature effectson glucose uptake in marine waters, Takahashiand Ichimura (24) found a range of temperaturesensitivity as measured by Arrhenius plots of logVmax versus the reciprocal of absolute tempera-ture. Surface waters and mixed-water massesgave the highest activation energies, a mean of21,900 + 1,500 cal (91,600 + 6,300 J). Lowermean values were found in deep, stable waters(10,600 ± 1,000 cal [44,300 ± 4,200 J]). Hamiltonet al. (12) found values of 34,200 (143,000 J) and26,800 cal (112,200 J) for the activation energiesof Vmax for glucose uptake in two cultured ma-rine bacteria. In view of these data, it is neces-sary to control and state carefully the tempera-ture under which activity measurements aremade. Given, say, an activation energy of 21,900cal (91,600 J), a 10°C difference in temperaturewould account for a fivefold variation in thespecific activity index of a natural population byits effect on uptake rate. Clearly, seasonal vari-ations in bacterial specific activity will be afunction oftemperature as well as other environ-mental or physiological factors.

MATERLA1S AND METHODSBacteria were counted with the AODC method of

Hobbie et al. (13), which uses Nuclepore filterscounterstained with irgalan black. An AO SpencerFluorestar epifluorescent microscope was fitted witha ruled ocular micrometer, and the bacteria in ran-domly selected fields were counted until at least 200were accumulated in the tally. In our hands betterresults were obtained by increasing the staining timeto 30 min and by using an irgalan black-counterstainedand oiled (type A; refraction index, 1.51) membranefilter (Millipore Corp.) as backing for the Nucleporefilter.

Bacterial activity was measured with the use of `4C-uniformly labeled organic solutes, employing the ki-netic approach of Wright and Hobbie (most recentlydescribed in 27) and the tracer approach of Azam andHolm-Hansen (2) to measure turnover rates. Enrich-ment experiments (direct uptake approach) involvedthe addition of labeled and unlabeled substrate tonatural water incubated in sterile biological oxygendemand bottles, under dark conditions. Plate countswere performed with 0.1-ml spread plates, using themedium of Murcheleno and Brown (18), dilutingwhere necessary with sterile seawater.

RESULTSV,,,-kinetic approach. Figure 1 and Table

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1 present data relating bacteria numbers andVmax specific activity for ["4C]glucose total up-take (assimilated plus respired) as measured ata 1-m depth along a transect from well up theEssex estuary (420 39' lat., 70° 45' long.) to a

station 14 km offshore in the Gulf of Maine. Thetransect involves a profound change in marinehabitats, from a shallow, salt-marsh-borderedtidal river to offshore waters 46 m in depth.Changes in bacteria numbers and specific activ-ity index suggest that the heterotrophic bacteriaare not uniformly active and, in fact, reflectphysiological differences that may well corre-spond with the habitat changes.

Figure 2 and Table 2 present total glucoseVmax specific activity and bacteria counts froma vertical profile taken at the 14-km-offshore

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station 9 days after the horizontal series. Again,major changes in specific activity accompanyhabitat differences, this time vertical location inthe water column.Turnover rate-tracer approach. The

simplicity of this approach facilitates the collec-tion ofmuch more data in a given period of time.Our practice has been to add ['4C]glutamate ata concentration of0.3 ,ug liter-' to a single sampleand a blank (to correct for background andisotope adsorption) and measure assimilation

only. Figure 3 represents one set of a monthlymonitoring series sampling from two fixed points3.5 km apart in the Essex estuary during a

complete flood tide. The salinity of the upriverstation (Fig. 3A) at high tide overlaps the salinityof the downriver station (Fig. 3B) at low tide;

FIG. 1. Bacteria numbers (clear) and Vma, specific activity index (10- 2 micrograms per cell per hour) forglucose uptake (stippled) at 1-m depth along a transect from within the Essex estuary to a station 14 kmoffshore, 4 August 1976.

TABLE 1. Data from horizontal profile from 1r-m depth along a transect from within the Essex estuary to astation 14 km offshore, 4 August 1976'

Station location and ..Secchi AODC V,.~glucose Vm.. sp act in-stationdeptahin and Salinity (%o) Temp (°C) dex (ug cell-' rbdepth ~~~~~~~depth (in) (xlO' nml-') (ug liter- h- ) h-1 10-12)

14 km out; 46 m 33.3 17.0 9 2.02 0.016 8.0 0.9010 km out; 34 m 33.3 17.2 9.5 2.26 0.040 18 0.867 km out; 36 m 32.3 16.9 6.5 2.01 0.22 110 0.904 km out; 28 m 32.0 17.3 4.5 3.27 0.16 50 0.991.5 km out; 7 m 32.0 17.3 4.5 4.94 0.75 150 0.99Estuary mouth; 8 m 32.0 18.5 2.5 5.07 1.31 262 0.961.5 km in; 7 m 31.6 20.3 1.5 4.05 0.96 240 0.993 km in; 3 m 29.8 20.7 2.5 8.21 0.87 160 0.954 km in; 2 m 28.0 20.9 1 8.29 0.81 98 0.87

Incubation temperature, 18°C."Correlation coefficient for linearity of V,,,,m calculation.

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155 10Specific Activity Index

FIG. 2. Bacteria numbers (clear) and V,,,,, specific activity indeX (1012 micrograms per cell per hour) forglucose uptake (stippled) from a vertical profile at a station 14 km offshore in the Gulf of Maine, 13 August1976.

TABLE 2. Data from a vertical profile at a station 14 km off Cape Ann in the Gulf ofMaine, 13 August1976a

AODC (x106 Plate count V,, glucose (,ug V. sp act indexcDepth (in) Salinity (%o) Temp ('C) ni'(FWiP)lte'h) (pg cell'I h'I r(CFUml-)liter-h-') l1-12)

1 31.9 18.6 1.87 6,120 0.053 28 0.995 33.7 16.2 1.59 2,140 0.018 11.3 0.9710 33.8 14.8 1.75 2,240 0.006 3.4 0.9115 34.0 10.0 0.66 1,120 0.0038 5.7 0.9820 34.0 9.4 0.61 540 0.0017 2.8 0.9125 34.1 9.2 0.76 330 0.0026 3.4 0.8830 34.1 9.0 0.79 220 0.0027 3.4 0.9635 34.1 8.8 0.76 170 0.0028 3.7 0.9740 34.2 8.7 0.72 240 0.0040 5.5 0.91

aIncubation temperature for glucose uptake, 13'C.b CFU, Colony-fonning units.c Correlation coefficient for linearity of Vm,. calculation.

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8-

0 1 2 3 4leers After Lew Tide

FIG. 3. Bacteria numbers (clear) and turnover ratespecific activity index (10' per hour per cell liters)for glutamate uptake (solid) of surface waters takenfrom fixed points in the Essex estuary during floodtide. (A) Data from a station 4 km upriver, 14 Novem-ber 1977: incubation temp, 5°C; natural tempera-ture range, 5 to 7°C; salinity range, 4.7 to28.8%o. (B) Data from station 1.5 km upriver, 15 No-vember 1977: incubation temperature, 7°C; naturaltemperature range, 5 to 8°C; salinity range, 27.6 to30.0%o.

other studies of the horizontal tidal range of thisriver have shown that the low tide-high tideexcursion of water from the downriver stationdoes in fact carry it to the upriver station. Figure3 indicates a midestuary peak in bacteria num-bers, with a somewhat erratic pattern of specificactivity. Figure 4 shows results from an entirelydifferent kind of estuary, the Merrimack River.It is a much larger river system, heavily polluted,and lacks the extensive salt marshes that char-acterize the Essex estuary. The data indicatehighest specific activity and numbers in thefreshwater portion of the estuary, a distinctivelydifferent pattern from the Essex, as shown inFig. 1 and 3. Moving downriver, brackish con-

ditions mark a drastic drop in specific activity,whereas numbers drop only slightly. Anotherway of using this approach is shown in Fig. 5,where both numbers and specific activity fromthe same run are fractionated with the use ofNuclepore filters. The polluted freshwater bac-teria are much larger in size than those in saline

waters, probably explaining some of the greatdifference in specific activity.Direct uptake measurement. Figure 6

shows the results of incubating replicate wintersamples of Essex estuary water at 2°C (the nat-ural temperature) and 15°C for 48 h, enrichedwith 100 or 300 ,ug of glucose liter-'. The patternin the 2°C sample is a substantial increase inspecific activity with little increase in numbers;with the higher incubation temperature, specificactivity increases more rapidly and then declinesdue to exhaustion of labeled substrate, whereasnumbers increase fourfold.

In another enrichment experiment, severalconcentrations of "C-labeled glucose were usedover 48 h with a midsummer water sample fromthe inlet of Essex Bay. Results (Table 3) indicatethe following: (i) a threefold increase in directcounts; (ii) exponential increase in the use ofglucose, with an increase in specific activity oftwo orders of magnitude; (iii) no significant in-fluence of varying glucose concentration on di-rect counts; (iv) "viable count" increases thatindicate definite dependence on glucose concen-

tration as well as a pattern of exponential in-crease. These data, and those of Fig. 5, clearlyshow pronounced increases in specific activity(and "viability") without large increases in num-bers of bacteria. The higher values for activityindex from the summer sample enriched withglucose are similar in magnitude to Vmax-per-cellvalues reported by Hamilton et al. (12) for sev-

eral strains of chemostat-isolated and -grownmarine bacteria. It seems likely that towards theend of the incubation time the enriched popu-lation was largely made up of active cells at closeto maximum metabolic activity for the temper-ature conditions.

DISCUSSIONThe main purpose of the data presented is to

indicate the usefulness of looking at hetero-trophic activity with a quantitative conventionsuch as the specific activity index. There are

some problems with this approach: sizes of bac-teria vary, temperature differences pose prob-lems of comparison, and three different indexesare offered which are not interconvertible. Yetthe data show substantial variations in bacterialactivity, variations which indicate some interest-ing patterns. For example, one of the most intri-guing questions about the coastal bacteria is: arethey largely a population that emerges fromestuarine inlets and gradually dies out as it ismixed with increasingly nutrient-poor water, or

are they an indigenous population directly re-

flecting the nutrient quality of the water inwhich they are entrained? For that matter, are

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SPECIFIC ACTIVITY IN BACTERIA OF NATURAL WATERS 303

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Distance Upriver (ki)FIG. 4. Bacteria numbers (clear) and turnover rate specific activity index (10-6 per hour per cell liters) for

glutamate uptake (stippled) from the Merrimack River estuary, 1-m depth, 21 November 1977. Values inparentheses: salinity in parts per thousand. Temperature range, 6.2 to 8°C; incubation temperature, 7°C.

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listauce Upriver (1in)FIG. 5. Bacteria numbers and turnover rate spe-

cific activity index (10-6 per hour per ceU liters) fromseveral stations along the Merrimack River estuary.Numbers and heterotrophic activity were fractionedusing Nuclepore filters of different pore sizes. Envi-ronmental data and stations as for Fig. 4.

the estuarine bacteria largely the result of ad-mixture from the salt marsh and estuarine sed-iments?Two recent papers by Novitsky and Morita

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FIG. 6. Bacteria numbers (clear) and direct uptakespecific activity index (10-12 micrograms per cell perhour) for glucose uptake (stippled) in Essex estuarywater enriched with 100 pg (2°C incubation) or 300pg (15°C incubation) ofglucose liter-'.

(19, 20) throw some light on these questions.The authors describe the transition of a labora-tory population of a Vibrio species during star-

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vation and subsequent reintroduction into nu-

trient-rich culture. Both size and endogenousrespiration are greatly reduced during starvationbut are restored when nutrients are once againprovided. From the present study, Fig. 6 andTable 3 show a pronounced increase in specificactivity by the natural bacteria in response toenrichment. In some enrichment experiments,there has also been a measurable shift frompredominantly coccoid to larger rod-shaped bac-teria during the incubation. Figures 1 and 2suggest the reverse process-as bacteria encoun-ter changes in the nutrient content of marinewaters, moving from richer to poorer nutrientenvironments, heterotrophic activity per cell de-clines rapidly. It appears, then, that althoughbacteria numbers may be related in general tothe nutrient quality of the water (or, more pre-

cisely, the water in which they originate), spe-

cific activity is a better indicator of the physio-logical state and metabolic role of the bacteriain direct response to organic nutrients. For ex-

ample, Fig. 4 and 5 show a highly active pollu-tion-related population of large freshwater bac-teria which, when diluted with seawater,changes or disappears and is replaced with a

much-lower-activity marine population domi-nated by the smaller cells (0.2 to 0.6 ,sm). In thetransition, total numbers do not change very

much.The specific activity index does not, of course,

TABLE 3. Enrichment of seawater with varyii9 Aug

distinguish between active and inactive bacteria.As yet there are no unequivocal methods foraccomplishing this. A few studies using labeledorganic solutes and autoradiography have showna range of percent active bacteria usually over

30% and as high as 100% (6, 11). These measure-

ments have used unnaturally high concentra-tions of substrate and hence are difficult to in-terpret. Other methods for detecting the "ac-tive" bacteria have been suggested based on

autoradiography with 33P (7) or on the presence

of a functioning electron transport system (16).These methods also indicate a variable but highproportion of active bacteria, by comparisonwith direct counts. The problem with all ofthesemethods is, of course, quantification. How activeare the active bacteria?The data presented in this paper support the

hypothesis expressed by several workers (20,23a) that the marine bacteria are adapted toconditions of nutrient starvation by becomingrelatively inactive, or "dormant," existing forperhaps weeks or months in a reversible physi-ological condition that reflects the availability oforganic nutrients. More measurements of spe-

cific activity, perhaps in conjunction with auto-radiographic data, should help to explain howimportant and widespread this phenomenon is.

ACKNOWLEDGMENTSI am grateful to Rebecca Buck for technical assistance in

many phases of this work.

ng concentrations of labeled glucose, Essex inlet,,ust 1976"

AODC (x 106 Plate count (CFU Cumulative amt Mean 12-h rate Direct uptake spSample AODC)(x nm11 lo-4)b ie' gltr act index (ILgampeml-') ml-' lo-4) used ( liter-) (Ag liter-' h-l) cel-' h-' 10-12)

Control (no added substrate)Oh 2.78 1.3312 h 2.9424 h . 3.26 5.2036h 6.1448 h 9.31 21.1

Glucose, 250 ,ug/liter addedOh 2.78 1.3312 h 3.06 1.5 0.13 42.424 h 3.72 6.26 15.8 1.2 32336 h 6.29 239 18.6 2,95748 h 9.44 32.6 243 0.40 42

.4Glucose, 500 ug/liter added

Oh 2.78 1.3312 h 2.66 2.1 0.18 67.724 h 3.25 4.84 18.1 1.5 46236 h 6.00 505 40.6 6,76748 h 8.82 43.0 434

Glucose, 1,000 iLg/liter addedOh 2.78 1.3312 h 2.81 3.8 0.31 11024 h 3.66 6.90 26.3 1.9 5.936 h 7.29 716 57.4 7,87448 h 9.87 69.2 956 20.1 2,036' Salinity, 32.1%o; incubation temperature, 17°C.^CFU, Colony-forming units.

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This material is based on research supported by the Na-tional Science Foundation, grants DES 73-6650 A02 and OCE77-19446.

LITERATURE CITED

1. Azam, F., and R. E. Hodson. 1977. Size distribution andactivity of marine microheterotrophs. Limnol. Ocean-ogr. 22:492-501.

2. Azam, F., and 0. Holm-Hansen. 1973. Use of tritiatedsubstrates in the study of heterotrophy in sea water.Mar. Biol. 23:191-196.

3. Blanchard, F. A., I. T. Takahashi, and H. C. Alex-ander. 1976. Biodegradability of ['4C]methylcelluloseby active sludge. Appl. Environ. Microbiol. 32:557-560.

4. Bowden, W. B. 1977. Comparison of two direct-counttechniques for enumerating aquatic bacteria. Appl. En-viron. Microbiol. 33:1229-1232.

5. Crawford, C. C., J. E. Hobbie, and K. L. Webb. 1974.The utilization of dissolved free amino acids by estua-rine microorganisms. Ecology 55:551-563.

6. Daley, R. J. Direct epifluorescence enumeration of nativeaquatic bacteria: uses, limitations and comparative ac-curacy. In Native aquatic bacteria; enumeration, activ-ity and ecology. Proceedings, A.S.T.M. Symposium, 25June 1977, Minneapolis, in press.

7. Faust, M. A., and D. L Correll. 1977. Autoradiographicstudy to detect metabolically active phytoplankton andbacteria in the Rhode River Estuary. Mar. Biol.41:293-305.

8. Ferguson, R. L., and P. Rublee. 1976. Contribution ofbacteria to standing crop of coastal plankton. Limnol.Oceanogr. 21:141-145.

9. Fry, J. C., and A. M. Ramsay. 1977. Changes in theactivity of epiphytic bacteria of Elodea canadensis andChara vulgaris following treatment with herbicide pa-raquat. Limnol. Oceanogr. 22:556-562.

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