A study of the distribution and activity of micro-organisms in ocean waters

A STUDY OF THE DISTRIBUTION AND ACTIVITY OF MICROORGANISMS IN OCEAN WATER1

John E. Hobbie, Department of Zoology, North Carolina State University, Raleigh

Osmund Holm-Hansen, Scripps Institution of Oceanography, La Jolla, California 92037

Theodore T. Packard, Department of Oceanography, University of Washington, Seattle 98195

Lawrence R. Pomeroy, Department of Zoology, University of Georgia, Athens 30601

Raymond W. Sheldon, Marine Ecology Laboratory, Bedford Institute, Dartmouth, Nova Scotia

James P. Thomaq2 Department of Zoology, University of Georgia, Athens 30601

and

William J. Wiebe Department of Microbiology, University of Georgia, Athens 30601

ABSTR4CT

Estimates of living mass and respiratory rate were made by several methods on large water samples taken at several depths at each of two ocean stations. Total microbial biomass estimates based on ATP determinations gave reasonable results in relation to phytoplankton biomass, total particulate organic carbon, and total particulate volume as determined by an electronic particle counter. Respiratory rate estimates based on 0, uptake, electron transport system activity, and ATP were in moderately good agreement in surface water but diverged widely in deep water. Heterotrophic uptake of specific organic compounds seems to be related to numbers of active bacteria and not related to total microbial respiration. Photosynthetic rates and release of dissolved photosynthate are reported for both stations.

INTRODUCTION

evaluation of the abundance and activity of microorganisms in the ocean is difficult because of their low concentration relative to the large amount of detrital material of similar size and density. A few techniques which utilize isotopic tracers permit direct measurements of metabolic activity in samples of ocean water. The

l This research was supported by several grants from the National Science Foundation and by U.S. Atomic Energy Commission Contract No. AT( 11-1) GEN 10, P.A. 20.

2 Present address: Department of Occanogra- phy, University of Washington, Seattle 98195.

widely used method of Stcemann Niclscn (1952) for measuring photosynthesis is one of the best known; the use of labeled organic materials to estimate potential hct- erotrophic activity is another (Wright and Hobbie 1966). With the exception of a few such as these, most methods for cval- uating either biomass or activity of microorganisms in the sea involve some sort of manipulation to concentrate the cells. Therefore, most methods are somewhat indirect and involve assumptions that have not always been tested. There is no as- surance that the several methods now used to estimate living mass and microbial metabolism are giving comparable results.

LIMNOLOGY AND OCEANOGRAPIIY 544 JULY 1972, v. 17(4)

DISTRIBUTION AND ACTIVITY OF MICROORGANISMS 545

We undertook a short oceanographic cruise to compare several of these methods on the same set of water samples. The region off Cape Hatteras is favorable for such a comparative study because diffcr- ent water types can be examined in a short interval of space and time. The water over the continental shelf and slope is rich and productive, while that of the western Sargasso Sea, 160 km away, is a relatively stable, stratified, and unproductive system, although not as oligotrophic as water near the center of the gyre.

We thank the Duke University Marine Laboratory for the use of RV Eastward on cruise E-41-69. Coulter Electronics, Inc., Hialeah, Florida, kindly loaned the model T particle counter for this study. We thank J. A. Marsh, Jr., J. Staley, R. Zingmark, L. W. Wood, D. Francisco, and L. Haas for their kind assistance during the cruise, G. Prcvost for technical assistance, and F’. Reid for the floristic analysis.

METHODS

The cruise plan

Two stations of contrasting water types were selected. Station 1 (36’25’ N, 74”43’ W) was over the continental slope north of Cape Hatteras. Station 2 (35’00’ N, 73 “00’ W) was in the extreme western Sargasso Sea, just beyond the eastern side of the Gulf Stream. Each station was oc- cupied for about 3 days. Water samples of 200 liters each were taken with a Gerard and Ewing ( 1961) sampler made of stainless steel and lined with vinyl. Wa- ter was removed from the sampler by gravity from a tap on the bottom equipped with a section of 12-mm Tygon tubing, and the samples divided among the inves- tigators. Water to be used for particulate carbon, pigments, and adenosine triphos- phatc (ATP) was prcfiltered through a 183-p nylon net directly into acid-cleaned borosilicate glass carboys. A portion of each sample was passed through a Dodson- Thomas reverse-flow concentrator ( Pome- roy and Johannes 1966, 1968). By the use of a two-stage concentrator, the particu-

late material from 200-300 liters was con-

centratcd into a final volume of 20-50 ml. The sample was maintained at in situ tem- peraturc during the 24-hr period of concentration. Each investigator carried out the methods for estimating living mass or metabolic rate with which he was espe- cially familiar; in most casts these were methods originated by or extensively modified by the investigator using them.

Estimates of living mass Bacterial mass

Freshly prcparcd concentrates were examined immediately at sea, using a Leitz phase/fluorescence microscope equipped with oil-immersion optics ( 1,000~ ) and special rubber mounting to minimize vi- bration. A drop of each concentrate was placed on a microscope slide and covered with a thin cover glass, a technique probably less damaging to naked protists than collection on a filter, and it permits obser- vation of motile organisms. Fluorescence using Acridine ,Orange as well as phase contrast microscopy was used to locate and count bacterial cells (Wood 1965). Marine bacteria, even at 1,000~ magnification, are difficult to distinguish by either technique, because they exhibit a high de- grce of pleomorphism, and many small particles in the sea naturally fluoresce at wavelengths in the green part of the spec- trum, similar to those emitted by stained bacteria. A combination of the two techniques permits a search for fluorcsccnt bodies followed by further verification of morphology and phase density.

Phytoplankton mass Portions of the original, unmodified wa-

ter samples were preserved in 4% Forma- lin, buffered with sodium borate to pH 8, for later counting by the Utermijhl ( 1931) method and subsequent estimation of mass ( Mullin et al. 1966; Helm-Hansen 1969).

Total microbial mass For ATP analysis l-2 liters of water

were filtered through 47-mm type HA Millipore membrane filters, The cellular

546 JOHN E. HOBBIE ET AL.

material was killed and cxtractcd as rapidly as possible by quickly removing the filter and submerging it in boiling Tris buffer. After heating it at 1OOC for 5 min, the tubes wcrc rcmovcd from the bath, cooled, and frozen at -20C until analyzed. The ATP content of the extract was dcter- mined by the luciferin-luciferase reaction (Holm-Hansen and Booth 1966). The ATP values were extrapolated to cellular organic carbon by multiplying pg ATP liter 1 by 250 (Hamilton and Helm-Hansen 1967; Helm-Hansen 1970).

Estimates of metabolic activity

ETS activity

The activity of the respiratory electron transport system ( ETS ) was measured by a tctrazolium reduction technique ( Curl and Sandberg 1961; Packard 1969; Pack- ard et al. 1971) on material filtcrcd from lo-30 liters of scawatcr, using Gehnan type A glass filters. The assay was stan- dardized by the method of Packard and Healy ( 1968)) although the absorbtivity method of Hirsch et al. (1963) works equally well. ETS activity at the in situ ocean temperatures was determined using the Arrhcnius equation and an energy of activation of 14 kcal mol- 1 (Packard 1969). Respiration rates in the region of high chlorophyll were equated with the ETS activity on the assumption that the respiratory control theory of Chance and Wil- liams (1956) does not apply to plants. Hackett ( 1964) gives evidence that plant mitochondria do not exhibit respiratory control. Below the region of high chlorophyll (below 100 m at station 1 and 200 m at station 2) hctcrotrophic dominance was presumed, and the respiratory control theory was applied. A factor of 5 was used in this region to reduce the respiratory capacity to a resting rate. Various factors have been suggested: 5 (Chance 1957), 6 (Chance and Williams 1955), 7 (Chance 1965), and 10 (Chance and Williams 1956). None of them is concerned with marinc microorganisms, The mean of the sug-

gcsted factors is 7, but Halcrow and Boyd

( 1967) presented evidence that spontanc- ously active organisms respire at 1.3 times the basal rate. Considering this, a mean estimate would bc ncarcr 5.

Oxygen uptake

Portions of the concentrates were placed in 4-ml respirometers in a dark incubator at the in situ tempcraturc of the sample. Changes in dissolved oxygen wcrc rc- corded continuously for at least 1 hr by means of oxygen electrodes (Carritt and Kanwishcr 1959). Significant amounts of living material are lost during conccntra- tion and a correction factor of about 3 is appropriate ( Holm-Hansen et al. 1970) ; the results are reported with this corrcc- tion. It is also assumed that concentration, as such, has no large cffcct on the rcspi- ratory rate; some cvidcncc for this has been rcportcd previously (Pomeroy and Johannes 1968). The respiromctcr was rinsed with 70% ethanol and a blank was run with membrane-filtcrcd scawatcr after each mcasurcmcnt. Oxygen demand of the Pt electrode produced a change of about 2% hrI. Therefore, a rate of change of less than 5% with a concentrate is of doubtful significance. This puts the lower limit of resolution of the method at about 0.5 ~1 02 liter-l day-l.

Heterotrophic potential

lMicrobia1 activity, as the uptake of low concentrations of specific organic compounds, was also measured. Subsamples of 5 ml of water or a dilution of the primary concentrate were incubated at cnvi- ronmental tcmpcratures for 4-6 hr with either glucosc-6-14C, prolinc-u-14C, or aspartic acid-u- ‘“C. Both the amount of the radioisotope retained by the bacteria and that respired were mcasurcd at four concentrations of substrate. Each conccntra- tion was replicated and one killed control was run for every radioisotope, giving a total of 9 flasks for each depth and radioisotope. Details of the method and calcu-

lations arc in Hobbie and Crawford (1969). The final result is an cstimatc of the


maximum velocity of uptake ( V, ) , which is a heterotrophic potential only. Bccausc the naturally prcscnt concentration of substrate is unknown, a true velocity of up-

take cannot be mcasurcd.

CO, uptake

Photosynthesis was cstimatcd by a l”C method that included measurement oE both the 14C incorporated in the phytoplankton and that released back into the water in dissolved organic products of photosynthesis. Incubation time was 6 hr ( dawn to noon or noon to sunset ) . Dc- tails of the proccdurcs arc given clscwhcrc ( Thomas 1971).

Respiration from ATP data

The microbial biomass as cstimatcd by ATP analyses has been further cxtrapo- lated to respiration rate (ml 02 liter-l yr-l) by multiplying by the factor 0.091. This factor has been obtained by taking a mean of respiratory rates of various bacteria, algae, and zooplankton as dctcrmined in laboratory cxpcriments (Coombs ct al. 1967; Laskcr 1970; Giesc 1962) and apply- ing tcmpcrature corrections. There is littlc information available on the relationship between tempcraturc and respiration rates for plant or animal cells living at various depths in the ocean. For our calculations, wc have assumed that the average rcspira- tiou rate on which WC have based our calculations [ 0.25 ~1 02 ( rug org C)-1 day-l at 22Cj will have a Qlo of 2.0 as used in the van’t Hoff equation.

Analyses of particulate matter

Total particle count and volume

Portions of water and concentrates wcrc analyzed immediately after collection with an electronic particle counter with a 14- channel pulse height analyzer ( Coultcr model T). The distribution of particles with cffcctivc diamctcrs from 0.5-128 p was determined, as well as the total particle volume. No methods specifically ap- plicablc to this instrument have yet been described, but in principle the methods

used did not differ significantly from those of Sheldon and Parsons ( 1967).

Particulate organic carbon

One to two liters of water were filtered at 25 mm of III; through precombusted 25- mm glass-fiber filters (Whatman GF/C) and the organic carbon was determined by infrared mcasurcmcnt of the CO2 libcr- atcd during wet combustion of the sample ( Mcnzcl and Vaccaro 1964; Helm-Hansen ct al. 1967).

Chlorophyll and pheophytin

One liter of the water sample was fil- tercd through a 25mm glass-fiber filter to which was added 2.0 ml of a 1% MgC03 suspension during filtration. The chlorophyll and pheophytin were determined fluorometrically (Holm-Hansen et al. 1965).

DNA

Scawatcr samples of from 0.1 to 4.0 li- tcrs were collected on 0.45-p membrane filters. Analysis was by the fluoromctric procedure utilizing 3,5-diaminobcnzoic acid ( Holm-Hansen ct al. 1968).

H y clrographic data

Tcmpcraturc, salinity, and oxygen were sampled with a separate series of Nanscn casts. The depths of the Nanscn bottles did not exactly correspond to those of the Gerard-Ewing samples that were used for other aspects of the study. The Nansen bottle depths reported arc calculated thcr- momctric depths. Water transparency was estimated from Secchi disk observations made each day within 1 hr of local appar- cnt noon,

RESULTS

Although station 1 was over the conti- ncntal slope, the water proved to be a laycrcd combination of warm Gulf Stream water and colder slope water. At station 2 the water structure and composition was typical of the western Sargasso Sea (Wtist 1957) ) with the permanent thermocline and the oxygen minimum at 600-800 m (Table 1).


TABLE 1. Hydrographic data at two stations in the western North Atlantic at which living mass and metabolism of microorganisms were studied in 1969

Thenno-

Sta. 1, 17 Nov

Thermo-

Sta. 2, 19 Nov

metric depth (In)

Temp (“C)

02 (mg liter-l)

metric depth (n1)

Temp (“C) (mg %er-l)

surf. 19.48 34.71 10.04 surf. 22.39 36.42 9.47 13 19.60 34.70 9.94 10 22.43 36.40 9.51 20 19.86 34.99 10.02 18 22.48 36.44 9.48 50 18.81 35.18 9.08 32 22.48 36.41 9.46

110 14.18 35.32 7.86 127 22.30 36.51 9.26 151 11.87 35.44 6.08 142 20.65 36.66 8.63 200 12.70 35.40 6.90 175 19.33 36.62 8.73 308 9.67 35.20 6.15 311 18.37 36.53 8.47 342 7.93 35.06 7.44 400 17.92 36.48 8.94 455 7.16 35.02 8.47 477 17.77 36.46 9.16 629 5.72 34.99 9.72 607 16.97 36.30 8.51 656 5.58 34.97 10.34 685 14.98 35.92 7.50

Estimates of living mass Data pertaining to the distribution and

activity of cells as well as various other estimates of the total particulate matter arc shown in Table 2 and Fig. 1.

The cellular organic carbon based on ATP values accounts for between 50 and 100% of the total organic carbon at depths between 4 and 100 m. At 700 m at station 1 the biomass-carbon value is only about

6% of the total organic carbon. Carbon values based on settled cell counts at 4, 10, and 100 m are a little less than that predicted by ATP.

At the 10-m depth at station 1 the settled cell counts indicated a value of 168 pg C liter-l, which is more than the carbon value obtained by direct combustion. Of this 168 ,ug C liter-l, 103 were recorded as monads ranging in diameter from 1 to

TABLE 2. Parameters of living mass and respiratory rate at two stations in the western North Atlantic

Phyto- Org Partic. plankton

(/a c c vol ATP DNA Chl n Pheo a

Depth (m/a Resp. (~10, day-l)

(ml liter-‘) (pLg liter-l) c:v liter-l) (lg liter -I) ETS 0, ATP

4 110 152 887 0.171 10 168 101 754 0.134 40 6 20 158 0.127

100 3 15 127 0.118 200 d 14 114 0.122 500 * 10 107 0.094 700 Q 12 * *

6 19 30 243

:o" 19 * 27 26 205 195 100 5 22 126 200 0.3 7 55 500 * 4 39 700 d 4 42

0.123 0.127 0.138 0.174 0.124 0.090 0.093

Station 1 518 5.2 420 6.2

63 2.7 28 1.2 16 0.9 6 0.6 3 0.5

Station 2 66 3.0 79 2.3 75 2.2 39 2.4

5 0.8 2 0.3 2 0.3

0.47 1.10 43.0 36.0 30.1 1.30 0.75 55.0 27.6 24.6 0.12 0.12 1.3 13.4 3.0 0.05 0.09 0.06 0.3 1.3 0.01 0.05 0.05 1.2 0.4 0.003 0.03 * 1.5 0.1 0.005 0.02 0.01 0.9 0.1

0.14 0.14 0.24 0.12 0.28 0.11 0.18 0.08 0.007 0.016 0.0009 0.006 0.0009 0.006

3.0 9.0 4.4 1.8 10.8 4.9 4.7 8.7 4.7 0.2 0.1 <ii

2.4 0.04

0.04 1.8 0.6 0.04 2.4 0.7

* Not sampled.

DISTRIRUTION AND ACTIVITY OF MICROORGANISMS 549

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I T.7 I T.7 STATION I STATION I - - . .

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FIG. 1. A comparison of some parameters of FIG. 1. A comparison of some parameters of living mass at two stations in the western North Atlantic, one over the continental slope and the second in the western Sargasso Sea, Symbols : 0 -volume ( ppm) ; o-total carbon (mg liter-l X 10) ; X-phytoplankton carbon (mg liter-’ x 10) ; &biomass carbon (mg liter-’ x 10) ; o- DNA, station 1 (,ug liter-’ x 2 X lo-‘), station 2 (fig liter-l X 1 X 10-l).

living mass at two stations in the western North Atlantic, one over the continental slope and the second in the western Sargasso Sea, Symbols : 0 -volume ( ppm) ; o-total carbon (mg liter-l X 10) ; X-phytoplankton carbon (mg liter-’ x 10) ; &biomass carbon (mg liter-l x 10) ; o- DNA, station 1 (,ug liter-l x 2 X lo-‘), station 2 (fig liter-l X 1 X 10-l).

15 JU. These objects, which are called monads in our analyses, look like cells under the microscope. Many have one or more flagellae, and the internal organelles usu- ally can be seen. As most are counted under high power ( 650~ ) , there is a greater possible error in extrapolating to concentrations per liter, We do not know whether the total carbon value obtained by combustion at the 10-m depth repre- sents a lack of homogeneity of the water

sample or whether our cell count is high bccausc of nonrandom distribution of monads on the microscope slide.

The particle counts from this cruise have been reported elsewhere (Sheldon et al. 1972, their Fig. 8, first and second se- rics from the left ). At station 1 the biomass is concentrated in several discrete size classes, which is characteristic of productive, coastal water. Station 2 shows the even distribution of particles characteristic of the open ocean. There is good agreement between these particle counts and the microscopic analyses with respect to the size distribution of organisms.

Microscopic examination at sea of fresh concentrates of particulate material, in- cluding ultraplankton, yields results that are less quantitative, because the cell numbers of most kinds of organisms are still too sparse. It does provide useful information on bacteria, small motile forms, organic detritus, and aggregates that cannot bc obtained by other means (Table 3). It is, for example, one of the most effective ways to observe living, metabolically ac- tivc bacteria. In those cases where active bacteria are present in sufficient numbers to be significant heterotrophic consumers, they will be seen in the fresh preparations and can be enumerated roughly.

Recognizable bacteria were observed in most samples (Table 3), but below the euphotic zone the numbers were very low, approaching the limit of sensitivity of the method (about 10” cells ml-l). The most notable aspect of bacterial distribution was the occurrence of active, motile bacteria at station 2 at 200 and 700 m. Motile bacteria are observed infrequently in fresh concentrates of particulate matter from open ocean water ( Wiebc, unpublished).

The spatial relations of organisms to aggregates and detritus are probably more nearly correct as seen in the fresh concentrates than in other types of preparations. Bacteria attached to particles of detritus or aggregates were noted, since there has been speculation concerning the relation of marine bacteria to detritus (Pomeroy and Johannes 1968). Even with Acridine


TAHLE 3. Microscopic observations at sea of freshly concentrated particulate material

DC@11

(In)

40 100 200 500 700

6 100 200 500 700

Bacteria No./ml of concentrate* Motile No. phytoplankton Dominant

cells free cells

with Chl/ml attached cells

aggrsgate prcscnt ( concentrate ) twt

Station 1 1.70 x loj <lo” - 1.7 x lOi floc 7.00 x 10R <lo” - 4.0 x 10” floc 1.60 x 10’ <lo” -- 2.0 x lo? floc 3.60 x 10’ <lo” - 7.0 x lo? floc

I: - $ floc and plate

Station 2 2.00 x 10’ <lo” -- 1.6 x 10’ floc and plate 2.50 x lo” <lO”

T 7.0 x lo2 floc and plate

4.00 x lo1 <lo3 8 plate 1.50 x 10’ <lO” - $ plate 3.50 x 10’ <lo” + $ plate

* All values were calculated using 10,000X concentration of particles. These values are the same as raw seawater counts/ml when divided by 10 (; 100 fields were counted at 1,000x magnification under phase contrast.

t See text for description of the two types of aggregates. 8 None seen.

Orange and phase microscopy, few recognizable attached bacteria wcrc found, and those were in the euphotic zone exclusively.

Other small, motile protists, mostly flag- ellates, were readily seen also. Viable autotrophs arc positively idcntificd by chlorophyll fluorescence. At both stations, numbers of viable phytoplankters de- creased rapidly with depth, but the numbers of cells were much higher in surface water at station 1 than at station 2. These observations, plus the lack of chlorophyll- bearing organisms in samples taken below the euphotic zone, suggest that the Gerard- Ewing sampler was not grossly contami- nated with surface water and that flushing of the sampler was satisfactory.

The distribution of types of aggregates at the two stations differed greatly with depth. Two generalized types of aggrc- gates are commonly observed in the ocean, flocculent masses and platelike bodies. Flocculent aggrcgatcs are composed of an array of separate particles bound together within a loose, gelatinous matrix. Living and dead phytoplankton often arc cmbed- dcd in these masses. Platelike aggregates consist of a homogeneous-appearing pla- nar matrix with irregular, angular edges and organic and inorganic fragments em-

bedded in or attached to the particle (see Gordon 1970a, b for illustrations), At station 1 flocculent particles (floes) predom- inated at all depths examined. At station 2 floes wcrc obscrvcd only above 200 m. In the deeper samples plates were abun- dant, their presence coinciding with the greatest discrcpancics bctwccn ETS activity and oxygen uptake.

Metabolic activity

Respiration of total microorganisms, in- cluding phytoplankton, the smaller zooplankton (primarily Protozoa), bacteria, and fungi, was estimated by three methods: the electron transport system (ETS ) method, the concentration and oxygen rcs- pirometry method ( 0,)) and the method of using the standing stock of ATP as a parameter of respiration (ATP). Agrce- mcnt of all three methods in the euphotic zone at both stations is good, but still lcavcs much to be desired. Differences of 2~ are common, and there is one differ- cnce of 10x ( Table 2). Below 200 m at both stations thcrc is wide divcrgencc of results, with ETS giving the lowest values and 02 giving the highest values.

Rates of uptake of labeled organic substrates proved to be very low (Table 4). When plotted against substrate conccntra-

D1STRIB’WTION AND ACTIVITY OF MICROORGANISMS 551

TAl3LE 4. Heterotrophic potential as estimated from the uptake of labeled organic compounds. The concentration factors of the concentrates are estimated (A) from initial and final water uol- umes, (B) from isotope uptake, and (C) from V,,,,, data on concentratecl and unconcentratecl samples

V Concn factor Depth

“) II x

(m) Substrate (AM&y-’

A B c

Station 1 10 glucose 0.006 212 35 19

aspartic 0.41 proline 0.009

100 glucose 0.0005 494 141 aspartic 0.01 58 proline 0.002G 164

Station 2 13 glucose 0.003 213 1 4

aspartic 0.05 10 48 proline 0.084 5 2

200 glucose 481 160 aspartic 4.35 481 proline 1.66 240

tion in a modified Linewcaver-Burk cqua- tion, the uptake will yield a straight line if the microorganisms are actively taking up the substrate (Wright and Hobbie 1966). Although estuarine and coastal wa- tcrs always give good results with very little scatter of points around the line of best fit, the experiments reported here, particularly with Sargasso Sea water, show low uptake and much scatter of the plotted points. Scatter may result from the low counts of substrate taken up. For cx- ample, in the glucose cxpcrimcnt at 10 m, station 1, only a few hundred counts per minute wcrc removed by the organisms (5-20 X 10” counts min-l were added). A difference of only 20 counts min-l due to experimental and counting errors would introduce a fair amount of scatter, Ac- cordingly, one to three points out of eight wcrc dropped to allow calculations to bc made.

The experiments with uptake of organic substrates sccmcd to offer an opportunity to test the efficiency of the concentration method to retain cells. Accordingly, the concentration factors based on the vol- umes of initial water samples and final

TAISLE 5. Photosynthetic proc-luction (in mg C m-” clay-‘) of particulate (P) and released soluble (S) organic matter at two stations in the western

North Atlantic

Depth ( 1” ) P S Totnl

Rel radiation

(%)

surf. 4

10 16 23

surf. 4

10 16 23

surf. 6

13 20 28

surf. G

13 20 28

surf. 5

11

Station 1, 17 Nov, 1145-1700 5.5 0.8 6.3 7.3 0.5 7.8 3.9 0.6 4.5 6.0 1.0 7.0 3.3 0.2 3.5

Station 1, 18 Nov, 0650-1210 11.3 1.5 12.8 15.8 1.0 16.8 10.4 0.7 11.1 18.0 0.9 18.9

3.3 0.3 3.6

Station 2, 19 Nov, 1140-l 720 1.8 1.0 2.8 1.4 0.9 2.3 3.5 0.4 3.9 1.0 0.3 1.3 1.2 0.2 1.4

Station 2, 20 Nov, 0630-7215 0.9 0.8 1.7 1.1 0.G 1.7 1.2 0.1 1.3 0.3 0.0 0.3 0.1 0.0 0.1

Station 2, 21 Nov, 1055-l 700 1.7 0.5 2.2 1.8 0.9 2.7 2.0 0.8 2.8

100 58 29 16

8

100 58 29 16

8

100 58 29 16

8

100 58 29 16

8

100 58 29

conccntratc (column A, Table 4) were compared with concentration factors de- rived from the uptake data. Column B, Table 4, is based on the ratio of the mean counts per minute taken up at all conccn- trntions of substrates after concentration to the avcragc taken up before conccn- tration. If uptake can bc described by Michaclis-Mcnten equations, then the only term that will bc affcctcd by the conccn- tration will bc V,,. This increase in VI,,, which will bc directly proportional to the concentration, will cause the counts per minute to increase in the same manner. Column C, Table 4, shows the ratios of the V,, of the concentrate to those of the natural water.

552 JOHN E. HOBBIJ3 ET AL.

Total photosynthetic rate is shown in Table 5. In the more productive slope water, loss of released soluble photosynthate is of little importance to the csti- mation of total photosynthesis, but the absolute amount released is as large as that relcascd by the populations of the Sargasso Sea which are subject to the stress of nutrient deficiency, In the latter case, loss of soluble materials is highly significant, even in experiments lasting only half a day.

DISCUSSION

The five estimates of living mass in the sea compared in Fig. 1 give remarkably similar results. Although we do not have sufficient replication to permit the calcu- lation of standard errors for each method and depth, it is evident that we could estimate the amount of living mass about equally well with any of the five. Al- though this is encouraging, it should be noted that two of the analysts, particulate volume and DNA, include in their values large amounts of nonliving material. These two analyses correlate with living mass, not because they are direct measures of it but for more obscure reasons, It is possible to estimate living mass directly with the Coulter counter by time-delay analyses (Strickland and Parsons 1968), but we performed only a single count on each sample, which is expressed as total particulate volume. The DNA method measures both the DNA in living organisms and a larger amount of residual DNA ( Helm-Hansen et al. 1968). Therefore, it is surprising that total DNA in the water column seems to be correlated with living mass even though most of it is not associ- ated with a living organism at the time of collection.

The volume of the particulate material at any depth in either profile was close to 10 times the weight of total particulate organic carbon. This 10: 1 ratio for fresh weight: organic carbon is typical of most microbial cells, However the detrital fraction in ocean water also contains inorganic material. The ratio of 1O:l indicates that

the amount of inorganic material at both of our stations was small relative to the organic fraction. It does not, however, indicate that all of the organic fraction was living.

The use of direct microscopic counts of bacteria as a means of estimating living bacterial mass has been successful in environments where bacterial numbers are high, such as soils (Babuik and Paul 1970). In the open ocean, bacterial numbers are so low that even with a concentration factor of 20,000, few recognizable bacteria were seen. However, if we assume that most metabolically active bacteria are in recognizable form and that bacteria are not selectively removed in the concentration process, then their direct enumeration at least tells us that bacteria are not present in sufficient numbers to constitute a significant fraction of the living mass or to contribute significantly to the respiratory processes we measured. A further note in confirmation of this is below.

The very low V, values for uptake of organic substrates are comparable to those found in arctic and subarctic lakes (Rodhe et al. 1966) and 3 to 4 orders of magnitude lower than rates found in estuaries and eutrophic lakes. The numbers of bacteria estimated by direct counting are also com- parably smaller than one would find in estuaries and cutrophic lakes. In contrast, the 200-m sample at station 2 had unex- pectedly high rates of uptake. Motile bacteria were found only in this sample and in the sample at 700 m. Microscopy sug- gests that where there are motile bacteria in significant numbers, uptake of organic substrates is accelerated. It also seems to verify the earlier evidence that bacteria and not phytoplankton are primarily responsible for the heterotrophic uptake of dissolved organic substrates. It would ap- pear that the activity measured by the uptake of labeled organic substrates is a small fraction of the tota respiration measured by respirometry, ATP, or ETS activity. Most of the respiration must be nonbacterial, or, if it is bacterial, it must


involve substrates not tested and bacteria not seen by us.

The substrate uptake experiments also have some bearing on this problem. In most casts concentration factors based on substrate uptake gave much lower values than those predicted from initial and final volume of water (Table 4). The one exception is the 200-m sample at station 2, where motile bacteria were present in significant numbers, and the concentration factors are in good agreement. Of course, nonmotile bacteria may be lost sclcctivcly in the process of concentration, or they may be too infrequent or metabolically inactive to give good results with the substrate-uptake method.

The three methods of estimating respiratory rate are in sufficiently good agreement in surface water at both stations that we can have confidence that our estimates are the correct order of magnitude. The results of the three methods diverge with depth to differences of as much as 90X. We do not know if the differences found between methods in deep or unproductive water are due to physiological differences in the organisms in these environments, as compared with productive surface water, or if they result from faulty assumptions in our methodology. The observed respiratory rates below 200 m are at the lower limit of sensitivity of the 02 method, and the values calculated may be too high for this reason.

The methods used do not permit us to differentiate between the respiration of autotrophs and heterotrophs, but some in- ferences are possible. Respiration of phytoplankton generally shows a relationship to the amount of release of soluble pho- tosynthatc. A high rate of loss implies nutrient deficiency or other stress, accom- panied by a high respiratory rate (Thomas 1964). The experiments of Steemann Niel- sen and Hansen (1959) suggest that our station 1 would be expected to have low phytoplankton respiration ( 10% of gross photosynthesis) and our station 2 would be expected to have high phytoplankton respiration ( 25% of gross photosynthesis ) ,

In both cases total respiration greatly ex- cecds these amounts, The difference is attributed to respiration of heterotrophic microorganisms, which consume a large part of the primary production rapidly.

The identity of those microorganisms principally responsible for the observed respiratory activity remains obscure. We have presented limited evidence that it is not primarily bacteria, for direct counts and uptake of specific dissolved organic substrates show increases that are not re- flected in 02 utilization, ETS activity, or ATP content. Conversely, samples that show high respiratory rate do not have high counts of bacteria or high rates of uptake of dissolved organic substrates. Identification of the organisms responsible for most of the respiratory activity is one of several steps that would help us inter- pret the large differences that we find between the three parameters of respiration that are compared here. Once their identity is known, it will be possible to learn the essential details of the biochem- ical and physiological processes that go into the total respiratory activity of microorganisms in the ocean and how these re- late to such parameters as ATP and ETS activity.

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A study of the distribution and activity of micro-organisms in ocean waters

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