APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1976, p. 360-367Copyright © 1976 American Society for Microbiology

Vol. 32, No. 3Printed in U.S.A.

Undecompressed Microbial Populations from the Deep Sea'H. J. JANNASCH,* C. 0. WIRSEN, AND C. D. TAYLOR

Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543

Received for publication 23 April 1976

Metabolic transformations of glutamate and Casamino Acids by natural mi-crobial populations collected from deep waters (1,600 to 3,100 m) were studied indecompressed and undecompressed samples. Pressure-retaining sampling/incu-bation vessels and appropriate subsampling techniques permitted time course

experiments. In all cases the metabolic activity in undecompressed samples waslower than it was when incubated at 1 atm. Surface water controls showed a

reduced activity upon compression. The processes involving substrate incorpora-tion into cell material were more pressure sensitive than was respiration. Thelow utilization of substrates, previously found by in situ incubations for up to 12months, was confirmed and demonstrated to consist of an initial phase ofactivity, in the range of 5 to 60 times lower than the controls, followed by a

stationary phase of virtually no substrate utilization. No barophilic growthresponse (higher rates at elevated pressure than at 1 atm) was recorded; allpopulations observed exhibited various degrees of barotolerance.

Extensive reviewing (7, 19, 20) of the litera-ture on microbial activities at high hydrostaticpressures draws attention to the fact that allexperimental studies in this area have beendone either with surface-borne bacteria or mi-crobial populations and isolates collected fromthe deep sea and held for some time at normalatmospheric pressure. ZoBell (19) characterizedthis problem of unavoidable decompression forlaboratory studies: "Although many bacteriafrom the deep sea survived [the retrieval proc-ess], this observation fails to prove that somebacteria, possibly the most sensitive ones, werenot destroyed by decompression. Answeringthis question may require the examination ofdeep sea bacteria at in situ pressures withoutsubjecting them to decompression." In addition,one may be tempted to speculate that suchhigh-pressure-adapted and decompression-sen-sitive bacteria, if they exist, would more likelybe active under deep-sea conditions than wouldnonadapted forms. The problem of their recov-ery can be viewed in analogy to that of psychro-philic bacteria, which have a maximum growthtemperature of about 20°C. Pure culture studiesavoiding decompression will ultimately be nec-essary to prove the existence of decompression-sensitive bacteria and their possible barophilicbehavior.

Studies on the rapid decompression of natu-ral populations of marine bacteria from a depthof 400 m only (13) showed some adverse effects

I Contribution no. 3755 of the Woods Hole OceanographicInstitution.

due to "pressure shock." According to ZoBell(20), however, "most stock cultures are not in-jured by being compressed to 1000 atm within 1or 2 minutes in nongaseous nutrient media andthen immediately decompressed at about thesame rate to 1 atm." The procedure of isolatingbacteria from the deep sea implies, of course,decompression and a possible preselection ofresistant organisms. Since the earlier work byZoBell and Morita (22) in the PhilippineTrench, it is known that substantial numbersof bacteria (most of them being psychrophilic)will grow on agar plates immediately after re-covery from depths of more than 10,000 m. Re-compression of these isolates to in situ pressureelicits different responses, which range fromcomplete growth inhibition to various degreesof growth reduction.The following questions remain: (i) what is

the in situ activity of those bacteria from thedeep sea that can be isolated at 1 atm; (ii) arethere bacteria in the deep sea that do not sur-vive retrieval and decompression; and (iii) if so,do they differ in their in situ activity from thosebacteria that do survive recovery?One way of attacking these questions is rep-

resented by in situ incubation experiments. Bylowering sterile media into the deep sea for insitu inoculation as well as incubation, de-compression is avoided. Control samples haveto be collected for parallel incubation at normalatmospheric pressure and in situ temperature(2 to 4°C). Such studies have been carried outover several years and with large numbers of

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replicas. A variety of substrates (agar, starch,gelatin, and 14C-labeled acetate, mannitol, glu-tamate, and Casamino Acids) were depositedand recovered with the aid of the research sub-

mersible ALVIN on permanent bottom stationsin the North Atlantic at a depth of 1,830 m (5)and in the Tongue of the Ocean at a depth of1,960 m, with incubation periods of severalweeks to 15 months. Surprisingly, the resultshave not been too different from those of analo-gous experiments done with surface-borne bac-teria reported earlier (3), i.e., a similar retarda-tion of growth and metabolism of the in situincubated natural microbial populations, or

pure and mixed cultures, as compared withcontrols incubated in the laboratory.These studies suffer from the distinct disad-

vantage, however, that rates must be deter-mined from single end point measurements.Rates are estimated on the assumption that theactivity determined is continuous and constantover the entire incubation period, a fact thatcannot be proven. Furthermore, large numbersof replicate experiments necessary for statisti-cal significance are difficult to obtain when de-pending upon diving opportunities with a sub-mersible. We decided, therefore, to construct a

bacteriological pressure-retaining sampler forthe retrieval of deep-sea samples in the absenceof decompression. To avoid transfer problems,the vessel is also used as a culture chamber.Any number of subsamples may be withdrawnduring time course experiments without affect-ing the pressure within the vessel. The firstinstrument to be used to a depth of 2,000 m wasbuilt and tested in 1973. The detailed descrip-tion of its operation (6) is principally the sameas that for a more recent instrument that can

be used for depths up to 6,000 m. The firstresults obtained with these instruments are de-scribed in the present report.

MATERIALS AND METHODS

Sampling. Two sampler/incubation vessels havebeen used for this study (Fig. 1), one for collectingwater samples to depths of up to 2,000 m and theother for depths up to 6,000 m. Prior to sampling,two free-floating pistons (Fig. 2, a and b) are in theiruppermost position, with sterile fresh water in thetwo sections of chamber B and a gas precharge inchamber C. When the sampler is lowered from theship to the depth of sampling, the intake valve (f) inthe upper end plate is opened by a messenger. As thesample enters the Teflon-coated chamber A, the twopistons move downward at a speed set by sterilefresh water in the upper section of chamber B, pass-ing through a small orifice adjusted by the set-screwc to a filling time of 15 min. This pressure-snubbingdevice prevents the generation of high shear forcesat the intake. Chamber C is precharged with nitro-

FIG. 1. Pressure-retaining sampler/incubationvessels. Left: for use at 200 atm, 316 stainless steel,intake mechanism replaced by subsampling unit;right: for use at 600 atm (i.e., sampling depth, 6,000meters), Nitronic 50 stainless steel, trigger deviceattached.

gen to assure a sufficiently large gas cushion at thefinal pressure. Precharge pressures are calculatedon the basis of the estimated final depth of samplingand are, for example, 64 and 102 atm for the twosamplers (Fig. 1) when operated at 2,000 and 6,000 mof depth , respectively. The gas accumulation cham-ber is vital for preventing any substantial losses ofhydrostatic pressure within chamber A due to smallleakages or volume changes by expansion of thevessel. A check valve closes chamber A (maximumvolume, 1 liter) when the filling is completed. Thesample is well enough insulated to prevent tempera-ture changes of more than a few degrees duringretrieval through warmer surface water and trans-fer into a shipboard refrigerator set at 3°C. Theactual minimum depth of sampling is determined bymeasuring the pressure within the sample. For in-cubation, the samplers are inverted and positionedover a magnetic stirrer (stirring bar d).

In the larger sampler (Fig. 1), capable of with-standing 600 atm (safety margin of 4.0), the trigger-ing level is affixed to the toggle valve located justprior to the snubbing orifice in the center section.For protection from contamination, the intake iscovered by a cap containing a small sterile filtermembrane. During lowering of the sampler, sterilewater occupying the space between the intake andsnubbing device is gradually compressed, causingsome seawater to pass through the filter. The trig-gering mechanism uncovers the intake nozzle justbefore the filling starts. This precaution has beenfound necessary in earlier work using a tracer orga-nism as an indicator of contamination from the un-

sterile surfaces of the sampling gear (4). Details

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362 JANNASCH, WIRSEN, AND TAYLOR

en ical Corp.; specific activities of 1.1 mCi/mg and 200u:rrQ mCi/mmol, respectively) was added to unlabeled

carrier solutions and introduced into the pressurizedr X/ -1 incubation vessels via the sterile transfer unit to a

final concentration and activity of about 5 ,ug/mlwW&\XX& and 0.005 ,Ci/ml, respectively. Due to the variable

degree of mixing during the introduction of a sub-strate sample, the initial concentrations are notidentical from experiment to experiment and areindividually indicated in the legends of the graphi-

!i~zwIlllll,llfcally presented data (Fig. 3 through 5).Control samples, collected in sterile Niskin sam-

A4,2t///,',mlers at the same time and depth as the undecom-pressed samples, were incubated in stoppered, 1-liter Erlenmeyer flasks at in situ temperatures. The

_l\_|\radiolabeled substrates were added prior to incuba-SA \ tion. The closed gas phase in these 1-atm controls

was kept as small as possible, never exceeding 10%of the sample volume, and was assumed not to affectthe CO2 measurements. The same is held for theexpected slight increase of the dissolved oxygen con-tent. In the original samples oxygen was measuredto be 290 to 300 ,uM or about 86 to 88% air saturation.The validity of the above assumptions was con-firmed by a separate experiment (Fig. 5).Under the conditions of the experiments, oxygen

limitation could not occur in the undecompressedsamples. Complete oxidation of the available sub-strate to CO2 would result in a reduction of the

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FIG. 2. Scheme ofsamplerlincubation vessels. (A) , 1.0 0|o oSample chamber (Teflon lined); (B) two sections ofa 0freshwater-filled chamber separated by a pressure- 1Ilsnubbing device; (C) precharged air cushion; (a) and 0.5 ° 0 300 atm(b) free-floating pistons; (c) set screw for pressure- i.-, . p ,-- , -7,rsnubbing orifice; (d) stirring bar; (e) subsampling 5 10 15 20unit; (I) intake valve. Check valve, toggle valve, andintake cover are not shown. DA YS

FIG. 3. Incorporation (0) and respiration (0) ofsuch as precharge calculations, design of intake glutamate (initial concentrations, 5.58 and 5.78 pg/cover, toggle valves, flow-snubbing orifice, etc. ml) of water samples taken at depths of 1,800 andwould go beyond the framework of this paper. The 3,000 m at the Bermuda transect station "MM"information is available upon request. (34°45'N, 66°30'W) during incubation at 1 atm (ini-A mixture of U-_4C-labeled amino acids or L-[U- tial concentration, 5.56 Mg/ml) and at in situ pres-

14C]glutamic acid (International Nuclear and Chem- sure and temperature (3.5°C).

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UNDECOMPRESSED MICROBIAL POPULATIONS 363

5 sample is decompressed through the appropriate

O _ 170 atm port for analytical purposes.-0 o - It should be noted that for a more efficient use of.5 /° our two pressure vessels for incubation primarily,

-------- we recently built a special pressure-retaining sam-_______________________________-' pling unit that concentrates a 3-liter sample during

.5 - filling to 15 ml by Nuclepore filtration. Concen-0 trated and undecompressed subsamples can be

o0 - taken and stored in transfer units for later inocula-.5 ° 1 atm tion into the prepressurized incubation vessels. By

this means larger numbers of samples can be taken.0 _ /.-*10 from various stations on a single cruise. The re-

C0°°-- 313atm quired storage/transfer units are equipped with5 ____ small gas accumulators to prevent pressure loss.

V ^i==. t t v Data obtained from this sampling-storage-incuba-1 3 5 7 10 12 tion procedure will be reported separately.

Analyses. Of each of the 12-ml subsamples takenDA YS after various intervals of incubation, 10 ml was fil-

4. Incorporation (a) and respiration (0) of tered through a 0.22-,um membrane filter (Millipore,zino Acids (initial concentrations, 4.04 and 1.4 Corp.) and washed with 2 volumes of chilled seawa-of water samples taken at depths of1,700 and ter to determine the portion of substrate incorpo-m at the North Atlantic station "DOS 2" rated into cell material and pools (incorporation).>'N, 69041'W) during incubation at 1 atm (ini- Following the procedure of Wirsen and Jannaschncentration, 5.0 pg/ml) and in situ pressure (16), duplicate 0.3-ml samples were used for measur-mperature (3.5°C). ing 14CO2 production (respiration). Since remaining

substrate and labeled dissolved intermediates werenot measured, the term substrate utilization refers

latm, 22 l otm, 4' to the total amount of substrate metabolized, i.e.,F F_O-0-the sum of incorporated and respired 14C-labeledVm V material. The radioactivity was counted using an

V 1< V / Intertechnique SL-20 scintillation spectrometer.LL Wf method.*~-X Quenching was corrected for by the channels ratio

AI. IS method.r 190atm, 220

0-00~-0

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190 atm, 4'

50 100 150 50

RESULTS

150-o-

100 150 200 335

HOURS

FIG. 5. Incorporation (-) and respiration (0) ofglutamate (initial concentration, 5.0 pg/ml) in sur-

face seawater collected near Woods Hole during incu-bation at two pressures and two temperatures. The 1-atm and 22°C experiment was conducted in the de-compressed pressure sampler (@, 0) as well as instoppered Erlenmeyer flasks (A, A).

oxygen concentration by no more than 50%.Subsampling. Subsamples (maximally 13 ml) can

be removed from chamber A and equal portions ofliquid medium can be introduced by attaching a

transfer chamber (Fig. 1 and 2e). Sterile seawater or

medium is contained in the transfer chamber andpassed into chamber A by moving the hand-crankedpiston. If a subsample is taken at the same time, no

outside pressure source is needed for the operation.Ifa subsample of 10 ml is taken without the simulta-neous addition of a liquid sample or compensationfrom an outside pressure source to chamber C, thepressure within the culture chamber will decreaseabout 3%. More details are given by Jannasch et al.(6). After detachment of the transfer unit, the sub-

The following data have been obtained onseveral separate cruises and geographical loca-tions as indicated. The temperature, hydro-static pressure, type of substrate used, and ini-tial concentration of the substrate are indicatedin the figures or their legends. Since the initialsubstrate concentrations vary (see above), thedata on the degree of substrate utilization aregiven in percentages.In most experiments, the amount of sub-

strate incorporated decreases after an initialpeak due, probably, to the release of labeledmaterial and products of autolysis. Identifica-tion of these materials is underway. For theestimation of rates and substrate utilization,only those data are used that were obtainedduring the period of increasing substrate incor-poration.

In the first experiment reported here (Fig. 3),water samples were taken at 1,800 and 3,000 mand incubated undecompressed as well as at 1atm after the addition of glutamate. After 9days of incubation at 180 atm, 3.5% of the ini-tial amount of substrate was incorporated, ascompared with a 34.5% utilization after 2 daysat 1 atm. The corresponding values for respira-

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364 JANNASCH, WIRSEN, AND TAYLOR

tion are 17.5% utilization after 9 days at 180atm, as compared with 48.2% after 3 days at 1atm. The estimated rate of substrate incorpora-tion was 51 times and that of respiration 11times slower at in situ pressure than at 1 atm.Total utilization of glutamate after 9 days at180 atm was about four times less than that at 1atm.When the same experiment was done with

water samples taken from a depth of 3,000 m,the percentage of substrate incorporated at 300atm reached a plateau of 1.8% in 16 days,whereas at 1 atm 28.5% was incorporated in 3days. The corresponding values for respirationwere 18.5% at 300 atm in 16 days and 38% at 1atm in 3 days. Accordingly, the estimated ratesfor substrate incorporation and respirationwere 64 and 11 times slower, respectively, at insitu pressure than at 1 atm. Total utilization ofglutamate in 16 days at 300 atm was about 3.3times less than at 1 atm.Another experiment was conducted with Cas-

amino Acids as the substrate (Fig. 4). Sampleswere taken at depths of 1,700 and 3,130 m. At170 atm, the amount of Casamino Acid incorpo-rated was 9.4% after 4 days and the amountrespired was 23% after 6 days. The correspond-ing data for 313 atm are 10.3% incorporationand 22% respiration after 6 days. A 1-atm con-trol, which was done only for the sample takenat a depth of 3,130 m, showed a maximumCasamino Acid incorporation of 21% in 5 daysand a maximum respiration of 47% in 8 days.The rates of substrate incorporation and respi-ration were 9 and 4.8 times slower, respec-tively, at 313 atm than that in the decom-pressed sample. Total utilization of CasaminoAcids after 6 days at 313 atm was about 2.1-foldless than that at 1 atm.The effect of hydrostatic pressure on sub-

strate incorporation relative to respiration maybe expressed by the ratio M/I: ratio of the totalamount of substrate incorporated plus that res-pired (i.e., metabolized [M]) to the totalamount incorporated (I), both expressed in mi-crograms per milliliter. The data for these ra-tios were obtained over the period of initialactive growth, i.e., prior to the decline of sub-strate incorporation. Table 1 shows an increasein the M/I ratios with increasing pressure forthe data presented in Fig. 3 and 4. In compari-son to the 1-atm controls, increased pressureresulted in a greater portion of total substrateto be respired than incorporated. The agree-ment between the 1-atm data is excellent. Inthe experiment using Casamino Acids, a gener-ally preferred substrate, a slightly lower M/I

TABLE 1.. Metabolic ratios obtained frompercentages of total substrate incorporated plus

respired (M, 100%o) over the percentage of substrateincorporated (I) by undecompressed and

decompressed natural populations of marinemicroorganisms a

Pressure (atm) Total substrate incor- M/Iporated(%180 16 6.2

1 47 2.1300 11 9.0

1 43 2.3313 29 3.4170 27 3.7

1 44 2.3

a Substrates: glutamateCompare Fig. 3 and 4.

and Casamino Acids.

ratio was obtained at 313 atm than at 170 atm,but the values are still significantly higherthan in the 1-atm control.There were two more experiments conducted,

one in the Venezuelan Basin with samples col-lected at 1,600 m and one in the North Atlanticwith samples from 2,600 m. The results areprincipally the same as those reported above,but the data are somewhat less complete andtherefore not included.

In most of these experiments, the incubationwas continued for as long as 5 weeks, with nosignificant changes in the measurements.When the vessels were brought to room temper-ature and decompressed, incorporation as wellas respiration activities increased sharply aftera brief lag.To complete the picture of combined pressure

and temperature effects, glutamate incorpora-tion and respiration of a surface seawater sam-ple (collected in December 1975 near WoodsHole) were measured at 1 atm as well as at 190atm and at 4°C as well as at 22°C. The datapresented in Fig. 5 show the distinct effect of a190-atm increase in pressure, which was, how-ever, less pronounced than the 18°C drop intemperature. Pressure primarily reduced incor-poration, whereas a low temperature resultedin a pronounced lag. In combination, both ef-fects are expressed when the sample was incu-bated at 190 atm and 4°C.

In the samples incubated at 1 atm, the per-centage of the substrate utilized (about 95%)was unaffected by the 18°C temperature differ-ence. In the samples incubated at 190 atm,substrate utilization decreased from 85% mea-sured at 220C to 74% at 40C.

In a parallel experiment, incorporation andrespiration at 1 atm and 220C were measured in

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UNDECOMPRESSED MICROBIAL POPULATIONS 365

1-liter Erlenmeyer flasks. These data (Fig. 5)were nearly identical with those obtained in thesampler/incubation chamber under the sameconditions. These results rule out possible ap-paratus effects as being responsible for the lowmaximum utilization of substrate in pressur-ized cultures (Fig. 3 through 5).

DISCUSSIONIn general, the data of this study confirm our

earlier observations (5) on decreased microbialactivities in the deep sea. Adding to this infor-mation, originally obtained by end point meas-urements after prolonged in situ incubation,the present time course experiments showedthe rates of activity not to be constant. Aninitial incubation period of several days to sev-eral weeks, in which the activity was roughly 5to 60 times slower than in the controls, wasfollowed by a stationary phase, a virtual arrestof any further activity even though about 80%of the available substrate remained unutilized.It is apparent that increased hydrostatic pres-sure and decreased temperature resulted in botha retardation and a reduction of substrate utili-zation.Undecompressed cultures have been kept

long beyond the initial phase of activity with-out observing any further change in theamounts of incorporated and respired labeledmaterial. Continued metabolism at low levelsof activity is likely although not detectable byour measurements. When, at the terminationof the experiments, the pressure was releasedand the temperature was raised, the metabolicactivity was found to increase or to "recover"after a brief lag. This response indicates theabsence of an irreversible inhibitory effect. Atthis time, the limited utilization of substrate atincreased pressure and low temperatures isunexplained and will be subject to continuedresearch. This will include measurements ofunutilized substrate remaining and determina-tion of possible dissolved labeled intermediates.More specific points of discussion are the fol-

lowing. (i) Decompressed natural populationsof microorganisms collected at depths in therange of 1,700 to 3,100 m showed a considerablyincreased metabolic activity relative to unde-compressed populations. Vice versa, the meta-bolic activity of surface-originated populationswas markedly reduced when incubated underpressures of the range indicated above. Fromthese observations it appears that populationsfrom deep water and those from surface watersbehave similarly. It must be noted, however,that it is not possible to differentiate between

different components of the natural populationsuntil pure cultures can be obtained. Further-more, since there is no reliable way at this timeto assess the number ofthe metabolically activecells in the natural population, we did not ex-press our data on a "per cell" basis. Experimen-tal work with undecompressed pure culturesappears to be necessary.

(ii) The processes involving substrate incor-poration were more pressure sensitive than wasrespiration, as reflected by the increased M/Iratio. This notion is reviewed in some detail byPope and Berger (10). Data of Paul and Morita(9) showed that hydrostatic pressure and lowtemperatures affected glutamate incorporationmore than did respiration in a psychrophilicmarine bacterium. Schwarz and Colwell (11)recently reported that, of the total substrateconsumed, respiration increased approximately22% in pressurized samples of deep-sea sedi-ment bacteria in comparison to 1-atm controls.These results were principally confirmed in ourrecent study (17) with a number of psychro-philic isolates. In some preliminary pure cul-ture experiments, we have tried to include via-ble cell counts in order to express the data on a"per cell" basis. The results appear to indicate ahigher amount of substrate metabolized per vi-able cell at elevated pressure than at 1 atm.Until such data can be verified by appropriatecounting techniques, we ascribe this result to adecrease in viable cell numbers that has oc-curred during decompression. Obviously, via-ble counts from decompressed natural popula-tions will be even less useful for expressing theM/I ratio on a per cell basis.

(iii) If "barophilic" behavior is defined byhigher rates of growth and metabolism at ele-vated pressure than at 1 atm, all responses ofnatural microbial populations observed in thisstudy can only be described as "barotolerant";i.e., the metabolic activities were reduced bythe applied pressures to a variable degree.These results cannot be taken, of course, asproof for the absence of barophilic organismsthat may escape detection within the gross re-actions measured. Again, work with undecom-pressed pure culture isolates appears indispen-sable.The existence of truly barophilic microorga-

nisms, in the above sense, was indicated in theearly work of ZoBell and Morita (22) on a sul-fate-reducing isolate from the deep sea. Seki etal. (14) found in one of two rubber bulbs (J.-Z.sampler) inoculated and incubated for 5 days ata depth of 5,200 m that bacterial growth on apeptone-yeast extract medium had occurred at

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366 JANNASCH, WIRSEN, AND TAYLOR

a higher rate than in a 1-atm control. No cul-ture was kept. There is no direct proof and somedoubt (18) that the rubber bulbs, due to a loss ofelasticity at high pressure, actually fill at thesame depth that they are triggered and opened.On the basis of our earlier results, we have

speculated (5) that the principal site of micro-bial activity in the deep sea might be the intes-tinal tract of benthic animals, where prevailinghigh nutrient levels could support a populationof pressure-adapted microorganisms. There areno measurements done yet on undecompressedgut samples. Our studies on decompressed gutsamples from deep-sea fish, molluscs, arthro-pods, and echinoderms have resulted in nomore than pronounced barotolerant responses,when studied under in situ pressure, amount-ing to a 10 to 90% reduction of the activityobserved at 1 atm (unpublished data). Schwarzet al. (12), however, reported no or little effectof 750 atm on the increase of viable cell countswhen incubating a diluted sample of gut con-tent of amphipods recovered from 7,000 m. TheCO2 production from starch was 2.9% higher atin situ pressure than at 1 atm, indicating aslight barophilic behavior. When these enrich-ments were decompressed and pure cultureswere isolated, they showed typical barotolerantbehavior, i.e., a reduction of activity in therange of 45 to 65% at 700 atm as compared with1 atm, agreeing with other reports on barotoler-ant bacteria (8, 21, 23).

Discussions of these and other data whichindicate relatively low rates of microbial activ-ity at high pressure, as compared with 1 atm,should not overlook the fact that in situ activi-ties are still substantial in proportion to theamount of organic matter reaching the deep seafloor by sedimentation and do obviously notlead to any abnormal accumulation of organicmaterials. Artificially added organic waste ma-terials, however, not readily available as foodfor the macrofauna, may decompose at a consid-erably slower rate in the deep sea than in shal-low water.There is, at the present stage of this re-

search, an interesting discrepancy in the dataon the behavior of microorganisms in the deepsea versus observations made on benthic ani-mals. Some of the latter have been photograph-ically recorded to respond relatively quickly tosubmerged bait, locating and consuming itwithin periods from several hours to a few days(1, 2). Although the only in situ measurementof a metabolic process (oxygen consumption) todate on deep sea fish (15) resulted in relativelyslow rates, R. Turner (personal communica-tion) observed trapped specimens of benthic

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bivalves and arthropods in cages of small meshsize, implying a relatively high rate of growthwithin known time periods. Although stillqualitative, such observations indicate a mostintriguing variance to the strictly microbiologi-cal data. The apparent inconsistency might beresolved on the level of competition betweenmicroorganisms and higher forms of life underthe particular conditions of food distribution inthe deep sea.

ACKNOWLEDGMENTSWe gratefully acknowledge the engineering assistance of

K. W. Doherty and the laboratory assistance of S. J. Moly-neaux.

This work was supported by research grants DES75-15017 and OCE75-21278 from the National Science Founda-tion.

LITERATURE CITED1. Issacs, J. D. 1969. The nature of oceanic life. Sci. Am.

221:146-162.2. Issacs, J. D., and R. A. Schwarzlose. 1975. Active ani-

mals of the deep-sea floor. Sci. Am. 233:84-91.3. Jannasch, H. W., K. Eimhjellen, C. 0. Wirsen, and A.

Farmanfarmaian. 1971. Microbial degradation of or-ganic matter in the deep sea. Science 171:672-675.

4. Jannasch, H. W., and W. S. Maddux. 1967. A note onbacteriological sampling in sea water. J. Mar. Res.17:185-189.

5. Jannasch, H. W., and C. 0. Wirsen. 1973. Deep-seamicroorganisms: in situ response to nutrient enrich-ment. Science 180:641-643.

6. Jannasch, H. W., C. 0. Wirsen, and C. L. Winget. 1973.A bacteriological pressure-retaining deep-sea sam-pler and culture vessel. Deep-Sea Res. 20:661-664.

7. Morita, R. Y. 1972. Pressure 8.1 bacteria, fungi andblue-green algae, p. 1361-1388. In 0. Kinne (ed.),Marine ecology, vol. 1. Wiley-Interscience, New York.

8. Oppenheimer, C. H., and C. E. ZoBell. 1952. Thegrowth and viability of sixty-three species of marinebacteria as influenced by hydrostatic pressure. J.Mar. Res. XI:10-18.

9. Paul, K. L., and R. Y. Morita. 1971. Effects of hydro-static pressure and temperature on the uptake andrespiration of amino acids by a facultatively psychro-philic marine bacterium. J. Bacteriol. 108:835-843.

10. Pope, D. H., and L. R. Berger. 1973. Inhibition ofmetabolism by hydrostatic pressure: what limits mi-crobial growth? Arch. Mikrobiol. 93:367-370.

11. Schwarz, J. R., and R. R. Colwell. 1975. Heterotrophicactivity of deep-sea sediment bacteria. Appl. Micro-biol. 30:639-649.

12. Schwarz, J. R., A. A. Yayanos, and R. R. Colwell. 1976.Metabolic activities of the intestinal microflora of adeep-sea invertebrate. Appl. Environ. Microbiol.31:46-48.

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