APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1976, p. 360-367 Copyright © 1976 American Society for Microbiology
Vol. 32, No. 3 Printed in U.S.A.
Undecompressed Microbial Populations from the Deep Sea' H. J. JANNASCH,* C. 0. WIRSEN, AND C. D. TAYLOR Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543 Biology, of Department
Received for publication 23 April 1976
Metabolic transformations of glutamate and Casamino Acids by natural microbial populations collected from deep waters (1,600 to 3,100 m) were studied in decompressed and undecompressed samples. Pressure-retaining sampling/incubation vessels and appropriate subsampling techniques permitted time course experiments. In all cases the metabolic activity in undecompressed samples was lower than it was when incubated at 1 atm. Surface water controls showed a reduced activity upon compression. The processes involving substrate incorporation into cell material were more pressure sensitive than was respiration. The low utilization of substrates, previously found by in situ incubations for up to 12 months, was confirmed and demonstrated to consist of an initial phase of activity, in the range of 5 to 60 times lower than the controls, followed by a stationary phase of virtually no substrate utilization. No barophilic growth response (higher rates at elevated pressure than at 1 atm) was recorded; all populations observed exhibited various degrees of barotolerance.
Extensive reviewing (7, 19, 20) of the literature on microbial activities at high hydrostatic pressures draws attention to the fact that all experimental studies in this area have been done either with surface-borne bacteria or microbial populations and isolates collected from the deep sea and held for some time at normal atmospheric pressure. ZoBell (19) characterized this problem of unavoidable decompression for laboratory studies: "Although many bacteria from the deep sea survived [the retrieval process], this observation fails to prove that some bacteria, possibly the most sensitive ones, were not destroyed by decompression. Answering this question may require the examination of deep sea bacteria at in situ pressures without subjecting them to decompression." In addition, one may be tempted to speculate that such high-pressure-adapted and decompression-sensitive bacteria, if they exist, would more likely be active under deep-sea conditions than would nonadapted forms. The problem of their recovery can be viewed in analogy to that of psychrophilic bacteria, which have a maximum growth temperature of about 20°C. Pure culture studies avoiding decompression will ultimately be necessary to prove the existence of decompressionsensitive bacteria and their possible barophilic behavior. Studies on the rapid decompression of natural populations of marine bacteria from a depth of 400 m only (13) showed some adverse effects I Contribution no. 3755 of the Woods Hole Oceanographic Institution.
due to "pressure shock." According to ZoBell (20), however, "most stock cultures are not injured by being compressed to 1000 atm within 1 or 2 minutes in nongaseous nutrient media and then immediately decompressed at about the same rate to 1 atm." The procedure of isolating bacteria from the deep sea implies, of course, decompression and a possible preselection of resistant organisms. Since the earlier work by ZoBell and Morita (22) in the Philippine Trench, it is known that substantial numbers of bacteria (most of them being psychrophilic) will grow on agar plates immediately after recovery from depths of more than 10,000 m. Recompression of these isolates to in situ pressure elicits different responses, which range from complete growth inhibition to various degrees of growth reduction. The following questions remain: (i) what is the in situ activity of those bacteria from the deep sea that can be isolated at 1 atm; (ii) are there bacteria in the deep sea that do not survive retrieval and decompression; and (iii) if so, do they differ in their in situ activity from those bacteria that do survive recovery? One way of attacking these questions is represented by in situ incubation experiments. By lowering sterile media into the deep sea for in situ inoculation as well as incubation, decompression is avoided. Control samples have to be collected for parallel incubation at normal atmospheric pressure and in situ temperature (2 to 4°C). Such studies have been carried out over several years and with large numbers of
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replicas. A variety of substrates (agar, starch, gelatin, and 14C-labeled acetate, mannitol, glutamate, and Casamino Acids) were deposited and recovered with the aid of the research submersible ALVIN on permanent bottom stations in the North Atlantic at a depth of 1,830 m (5) and in the Tongue of the Ocean at a depth of 1,960 m, with incubation periods of several weeks to 15 months. Surprisingly, the results have not been too different from those of analogous experiments done with surface-borne bacteria reported earlier (3), i.e., a similar retardation of growth and metabolism of the in situ incubated natural microbial populations, or pure and mixed cultures, as compared with controls incubated in the laboratory. These studies suffer from the distinct disadvantage, however, that rates must be determined from single end point measurements. Rates are estimated on the assumption that the activity determined is continuous and constant over the entire incubation period, a fact that cannot be proven. Furthermore, large numbers of replicate experiments necessary for statistical significance are difficult to obtain when depending upon diving opportunities with a submersible. We decided, therefore, to construct a bacteriological pressure-retaining sampler for the retrieval of deep-sea samples in the absence of decompression. To avoid transfer problems, the vessel is also used as a culture chamber. Any number of subsamples may be withdrawn during time course experiments without affecting the pressure within the vessel. The first instrument to be used to a depth of 2,000 m was built and tested in 1973. The detailed description of its operation (6) is principally the same as that for a more recent instrument that can be used for depths up to 6,000 m. The first results obtained with these instruments are described in the present report. MATERIALS AND METHODS Sampling. Two sampler/incubation vessels have been used for this study (Fig. 1), one for collecting water samples to depths of up to 2,000 m and the other for depths up to 6,000 m. Prior to sampling, two free-floating pistons (Fig. 2, a and b) are in their uppermost position, with sterile fresh water in the two sections of chamber B and a gas precharge in chamber C. When the sampler is lowered from the ship to the depth of sampling, the intake valve (f) in the upper end plate is opened by a messenger. As the sample enters the Teflon-coated chamber A, the two pistons move downward at a speed set by sterile fresh water in the upper section of chamber B, passing through a small orifice adjusted by the set-screw c to a filling time of 15 min. This pressure-snubbing device prevents the generation of high shear forces at the intake. Chamber C is precharged with nitro-
FIG. 1. Pressure-retaining sampler/incubation vessels. 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, 6,000 meters), Nitronic 50 stainless steel, trigger device attached. to assure a sufficiently large gas cushion at the final pressure. Precharge pressures are calculated on the basis of the estimated final depth of sampling and are, for example, 64 and 102 atm for the two samplers (Fig. 1) when operated at 2,000 and 6,000 m of depth respectively. The gas accumulation chamber is vital for preventing any substantial losses of hydrostatic pressure within chamber A due to small leakages or volume changes by expansion of the vessel. A check valve closes chamber A (maximum volume, 1 liter) when the filling is completed. The sample is well enough insulated to prevent temperature changes of more than a few degrees during retrieval through warmer surface water and transfer into a shipboard refrigerator set at 3°C. The actual minimum depth of sampling is determined by measuring the pressure within the sample. For incubation, the samplers are inverted and positioned over a magnetic stirrer (stirring bar d). In the larger sampler (Fig. 1), capable of withstanding 600 atm (safety margin of 4.0), the triggering level is affixed to the toggle valve located just prior to the snubbing orifice in the center section. For protection from contamination, the intake is covered by a cap containing a small sterile filter membrane. During lowering of the sampler, sterile water occupying the space between the intake and snubbing device is gradually compressed, causing some seawater to pass through the filter. The triggering mechanism uncovers the intake nozzle just before the filling starts. This precaution has been found necessary in earlier work using a tracer organism as an indicator of contamination from the unsterile surfaces of the sampling gear (4). Details gen
,
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JANNASCH, WIRSEN, AND TAYLOR en u: rrQ
ical Corp.; specific activities of 1.1 mCi/mg and 200 mCi/mmol, respectively) was added to unlabeled carrier solutions and introduced into the pressurized incubation vessels via the sterile transfer unit to a final concentration and activity of about 5 ,ug/ml wW and 0.005 ,Ci/ml, respectively. Due to the variable &\XX& degree of mixing during the introduction of a substrate sample, the initial concentrations are not identical from experiment to experiment and are individually indicated in the legends of the graphi!i~ z wIlllll,llfcally presented data (Fig. 3 through 5). Control collected in sterile Niskin samA4mlers at thesamples, same time and depth as the undecom,2 t///,' , pressed samples, were incubated in stoppered, 1liter Erlenmeyer flasks at in situ temperatures. The \ _ | \radiolabeled substrates were added prior to incuba_l tion. The closed gas phase in these 1-atm controls SA \ was kept as small as possible, never exceeding 10% of the sample volume, and was assumed not to affect the CO2 measurements. The same is held for the expected slight increase of the dissolved oxygen content. In the original samples oxygen was measured to be 290 to 300 ,uM or about 86 to 88% air saturation. The validity of the above assumptions was confirmed by a separate experiment (Fig. 5). Under the conditions of the experiments, oxygen limitation could not occur in the undecompressed samples. Complete oxidation of the available substrate to CO2 would result in a reduction of the r
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such as precharge calculations, design of intake cover, toggle valves, flow-snubbing orifice, etc. would go beyond the framework of this paper. The information is available upon request. A mixture of U-_4C-labeled amino acids or L-[U14C]glutamic acid (International Nuclear and Chem-
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DA YS FIG. 3. Incorporation (0) and respiration (0) of glutamate (initial concentrations, 5.58 and 5.78 pg/ ml) of water samples taken at depths of 1,800 and 3,000 m at the Bermuda transect station "MM" (34°45'N, 66°30'W) during incubation at 1 atm (initial concentration, 5.56 Mg/ml) and at in situ pressure and temperature (3.5°C).
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sample is decompressed through the appropriate port for analytical purposes. O_ o It should be noted that for a more efficient use of /° our two pressure vessels for incubation primarily, 0. .5 -------we recently built a special pressure-retaining sampling unit that concentrates a 3-liter sample during _______________________________-' 2. .5 to 15 ml by Nuclepore filtration. Concenfilling 0 N, trated and undecompressed subsamples can be 2.1o0 taken and stored in transfer units for later inocula1 atm tion into the prepressurized incubation vessels. By ° 1 .5 this means larger numbers of samples can be taken from various stations on a single cruise. The re1.1.0 _ /.-*10 C0°°-quired storage/transfer units are equipped with 313atm 0. 5 ____ small gas accumulators to prevent pressure loss. t v Data obtained from this sampling-storage-incuba^i==. t V tion procedure will be reported separately. 7 10 1 3 12 5 Analyses. Of each of the 12-ml subsamples taken DA YS after various intervals of incubation, 10 ml was filFIG. 4. Incorporation (a) and respiration (0) of tered through a 0.22-,um membrane filter (Millipore Casam,zino Acids (initial concentrations, 4.04 and 1.4 Corp.) and washed with 2 volumes of chilled seawapg/ml, of water samples taken at depths of1,700 and ter to determine the portion of substrate incorpo3,130 m at the North Atlantic station "DOS 2" rated into cell material and pools (incorporation). (38019>'N, 69041'W) during incubation at 1 atm (ini- Following the procedure of Wirsen and Jannasch tial concentration, 5.0 pg/ml) and in situ pressure (16), duplicate 0.3-ml samples were used for measurand temperature (3.5°C). ing 14CO2 production (respiration). Since remaining substrate and labeled dissolved intermediates were not measured, the term substrate utilization refers l otm, 4' latm, 22 to the total amount of substrate metabolized, i.e., the sum of incorporated and respired 14C-labeled F F_O-03 material. The radioactivity was counted using an Vm V / V 1< V Intertechnique SL-20 scintillation spectrometer. LL W was corrected for by the channels ratio *~-X Quenching f method. N,j A IS I. method. 1.
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FIG. 5. Incorporation (-) and respiration (0) of glutamate (initial concentration, 5.0 pg/ml) in surface seawater collected near Woods Hole during incubation at two pressures and two temperatures. The 1atm and 22°C experiment was conducted in the decompressed pressure sampler (@, 0) as well as in stoppered Erlenmeyer flasks (A, A).
concentration by no more than 50%. Subsampling. Subsamples (maximally 13 ml) can be removed from chamber A and equal portions of liquid medium can be introduced by attaching a transfer chamber (Fig. 1 and 2e). Sterile seawater or medium is contained in the transfer chamber and passed into chamber A by moving the hand-cranked piston. If a subsample is taken at the same time, no outside pressure source is needed for the operation. If a subsample of 10 ml is taken without the simultaneous addition of a liquid sample or compensation from an outside pressure source to chamber C, the pressure within the culture chamber will decrease about 3%. More details are given by Jannasch et al. (6). After detachment of the transfer unit, the suboxygen
The following data have been obtained on several separate cruises and geographical locations as indicated. The temperature, hydrostatic pressure, type of substrate used, and initial concentration of the substrate are indicated in the figures or their legends. Since the initial substrate concentrations vary (see above), the data on the degree of substrate utilization are given in
percentages.
In most experiments, the amount of substrate incorporated decreases after an initial peak due, probably, to the release of labeled material and products of autolysis. Identification of these materials is underway. For the estimation of rates and substrate utilization, only those data are used that were obtained during the period of increasing substrate incorporation. In the first experiment reported here (Fig. 3), water samples were taken at 1,800 and 3,000 m and incubated undecompressed as well as at 1 atm after the addition of glutamate. After 9 days of incubation at 180 atm, 3.5% of the initial amount of substrate was incorporated, as compared with a 34.5% utilization after 2 days at 1 atm. The corresponding values for respira-
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TABLE 1.. Metabolic ratios obtained from tion are 17.5% utilization after 9 days at 180 percentages of total substrate incorporated plus atm, as compared with 48.2% after 3 days at 1 atm. The estimated rate of substrate incorpora- respired (M, 100%o) over the percentage of substrate incorporated (I) by undecompressed and tion was 51 times and that of respiration 11 decompressed natural populations of marine times slower at in situ pressure than at 1 atm. microorganisms a Total utilization of glutamate after 9 days at 180 atm was about four times less than that at 1 Pressure (atm) Total substrate incorM/I porated(% atm. When the same experiment was done with 180 16 6.2 1 47 2.1 water samples taken from a depth of 3,000 m, 300 11 9.0 the percentage of substrate incorporated at 300 1 43 2.3 atm reached a plateau of 1.8% in 16 days, 313 29 3.4 whereas at 1 atm 28.5% was incorporated in 3 170 27 3.7 days. The corresponding values for respiration 1 44 2.3 were 18.5% at 300 atm in 16 days and 38% at 1 a Substrates: glutamate and Casamino Acids. atm in 3 days. Accordingly, the estimated rates for substrate incorporation and respiration Compare Fig. 3 and 4. were 64 and 11 times slower, respectively, at in situ pressure than at 1 atm. Total utilization of ratio was obtained at 313 atm than at 170 atm, glutamate in 16 days at 300 atm was about 3.3 but the values are still significantly higher than in the 1-atm control. times less than at 1 atm. Another experiment was conducted with CasThere were two more experiments conducted, amino Acids as the substrate (Fig. 4). Samples one in the Venezuelan Basin with samples colwere taken at depths of 1,700 and 3,130 m. At lected at 1,600 m and one in the North Atlantic 170 atm, the amount of Casamino Acid incorpo- with samples from 2,600 m. The results are rated was 9.4% after 4 days and the amount principally the same as those reported above, respired was 23% after 6 days. The correspond- but the data are somewhat less complete and ing data for 313 atm are 10.3% incorporation therefore not included. In most of these experiments, the incubation and 22% respiration after 6 days. A 1-atm control, which was done only for the sample taken was continued for as long as 5 weeks, with no at a depth of 3,130 m, showed a maximum significant changes in the measurements. Casamino Acid incorporation of 21% in 5 days When the vessels were brought to room temperand a maximum respiration of 47% in 8 days. ature and decompressed, incorporation as well The rates of substrate incorporation and respi- as respiration activities increased sharply after ration were 9 and 4.8 times slower, respec- a brief lag. To complete the picture of combined pressure tively, at 313 atm than that in the decompressed sample. Total utilization of Casamino and temperature effects, glutamate incorporaAcids after 6 days at 313 atm was about 2.1-fold tion and respiration of a surface seawater samless than that at 1 atm. ple (collected in December 1975 near Woods The effect of hydrostatic pressure on sub- Hole) were measured at 1 atm as well as at 190 strate incorporation relative to respiration may atm and at 4°C as well as at 22°C. The data be expressed by the ratio M/I: ratio of the total presented in Fig. 5 show the distinct effect of a amount of substrate incorporated plus that res- 190-atm increase in pressure, which was, howpired (i.e., metabolized [M]) to the total ever, less pronounced than the 18°C drop in amount incorporated (I), both expressed in mi- temperature. Pressure primarily reduced incorcrograms per milliliter. The data for these ra- poration, whereas a low temperature resulted tios were obtained over the period of initial in a pronounced lag. In combination, both efactive growth, i.e., prior to the decline of sub- fects are expressed when the sample was incustrate incorporation. Table 1 shows an increase bated at 190 atm and 4°C. in the M/I ratios with increasing pressure for In the samples incubated at 1 atm, the perthe data presented in Fig. 3 and 4. In compari- centage of the substrate utilized (about 95%) son to the 1-atm controls, increased pressure was unaffected by the 18°C temperature differresulted in a greater portion of total substrate ence. In the samples incubated at 190 atm, to be respired than incorporated. The agree- substrate utilization decreased from 85% meament between the 1-atm data is excellent. In sured at 220C to 74% at 40C. the experiment using Casamino Acids, a generIn a parallel experiment, incorporation and ally preferred substrate, a slightly lower M/I respiration at 1 atm and 220C were measured in
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1-liter Erlenmeyer flasks. These data (Fig. 5) were nearly identical with those obtained in the sampler/incubation chamber under the same conditions. These results rule out possible apparatus effects as being responsible for the low maximum utilization of substrate in pressurized cultures (Fig. 3 through 5). DISCUSSION In general, the data of this study confirm our earlier observations (5) on decreased microbial activities in the deep sea. Adding to this information, originally obtained by end point measurements after prolonged in situ incubation, the present time course experiments showed the rates of activity not to be constant. An initial incubation period of several days to several weeks, in which the activity was roughly 5 to 60 times slower than in the controls, was followed by a stationary phase, a virtual arrest of any further activity even though about 80% of the available substrate remained unutilized. It is apparent that increased hydrostatic pressure and decreased temperature resulted in both a retardation and a reduction of substrate utilization. Undecompressed cultures have been kept long beyond the initial phase of activity without observing any further change in the amounts of incorporated and respired labeled material. Continued metabolism at low levels of activity is likely although not detectable by our measurements. When, at the termination of the experiments, the pressure was released and the temperature was raised, the metabolic activity was found to increase or to "recover" after a brief lag. This response indicates the absence of an irreversible inhibitory effect. At this time, the limited utilization of substrate at increased pressure and low temperatures is unexplained and will be subject to continued research. This will include measurements of unutilized substrate remaining and determination of possible dissolved labeled intermediates. More specific points of discussion are the following. (i) Decompressed natural populations of microorganisms collected at depths in the range of 1,700 to 3,100 m showed a considerably increased metabolic activity relative to undecompressed populations. Vice versa, the metabolic activity of surface-originated populations was markedly reduced when incubated under pressures of the range indicated above. From these observations it appears that populations from deep water and those from surface waters behave similarly. It must be noted, however, that it is not possible to differentiate between
365
different components of the natural populations until pure cultures can be obtained. Furthermore, since there is no reliable way at this time to assess the number ofthe metabolically active cells in the natural population, we did not express our data on a "per cell" basis. Experimental work with undecompressed pure cultures appears to be necessary. (ii) The processes involving substrate incorporation were more pressure sensitive than was respiration, as reflected by the increased M/I ratio. This notion is reviewed in some detail by Pope and Berger (10). Data of Paul and Morita (9) showed that hydrostatic pressure and low temperatures affected glutamate incorporation more than did respiration in a psychrophilic marine bacterium. Schwarz and Colwell (11) recently reported that, of the total substrate consumed, respiration increased approximately 22% in pressurized samples of deep-sea sediment bacteria in comparison to 1-atm controls. These results were principally confirmed in our recent study (17) with a number of psychrophilic isolates. In some preliminary pure culture experiments, we have tried to include viable cell counts in order to express the data on a "per cell" basis. The results appear to indicate a higher amount of substrate metabolized per viable cell at elevated pressure than at 1 atm. Until such data can be verified by appropriate counting techniques, we ascribe this result to a decrease in viable cell numbers that has occurred during decompression. Obviously, viable counts from decompressed natural populations will be even less useful for expressing the M/I ratio on a per cell basis. (iii) If "barophilic" behavior is defined by higher rates of growth and metabolism at elevated pressure than at 1 atm, all responses of natural microbial populations observed in this study can only be described as "barotolerant"; i.e., the metabolic activities were reduced by the applied pressures to a variable degree. These results cannot be taken, of course, as proof for the absence of barophilic organisms that may escape detection within the gross reactions measured. Again, work with undecompressed pure culture isolates appears indispensable. The existence of truly barophilic microorganisms, in the above sense, was indicated in the early work of ZoBell and Morita (22) on a sulfate-reducing isolate from the deep sea. Seki et al. (14) found in one of two rubber bulbs (J.-Z. sampler) inoculated and incubated for 5 days at a depth of 5,200 m that bacterial growth on a peptone-yeast extract medium had occurred at
JANNASCH, WIRSEN, AND TAYLOR a higher rate than in a 1-atm control. No culture was kept. There is no direct proof and some doubt (18) that the rubber bulbs, due to a loss of elasticity at high pressure, actually fill at the same depth that they are triggered and opened. On the basis of our earlier results, we have speculated (5) that the principal site of microbial activity in the deep sea might be the intestinal tract of benthic animals, where prevailing high nutrient levels could support a population of pressure-adapted microorganisms. There are no measurements done yet on undecompressed gut samples. Our studies on decompressed gut samples from deep-sea fish, molluscs, arthropods, and echinoderms have resulted in no more than pronounced barotolerant responses, when studied under in situ pressure, amounting to a 10 to 90% reduction of the activity observed at 1 atm (unpublished data). Schwarz et al. (12), however, reported no or little effect of 750 atm on the increase of viable cell counts when incubating a diluted sample of gut content of amphipods recovered from 7,000 m. The CO2 production from starch was 2.9% higher at in situ pressure than at 1 atm, indicating a slight barophilic behavior. When these enrichments were decompressed and pure cultures were isolated, they showed typical barotolerant behavior, i.e., a reduction of activity in the range of 45 to 65% at 700 atm as compared with 1 atm, agreeing with other reports on barotolerant bacteria (8, 21, 23). Discussions of these and other data which indicate relatively low rates of microbial activity at high pressure, as compared with 1 atm, should not overlook the fact that in situ activities are still substantial in proportion to the amount of organic matter reaching the deep sea floor by sedimentation and do obviously not lead to any abnormal accumulation of organic materials. Artificially added organic waste materials, however, not readily available as food for the macrofauna, may decompose at a considerably slower rate in the deep sea than in shallow water. There is, at the present stage of this research, an interesting discrepancy in the data on the behavior of microorganisms in the deep sea versus observations made on benthic animals. Some of the latter have been photographically recorded to respond relatively quickly to submerged bait, locating and consuming it within periods from several hours to a few days (1, 2). Although the only in situ measurement of a metabolic process (oxygen consumption) to date on deep sea fish (15) resulted in relatively slow rates, R. Turner (personal communication) observed trapped specimens of benthic
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bivalves and arthropods in cages of small mesh size, implying a relatively high rate of growth within known time periods. Although still qualitative, such observations indicate a most intriguing variance to the strictly microbiological data. The apparent inconsistency might be resolved on the level of competition between microorganisms and higher forms of life under the particular conditions of food distribution in the deep sea. ACKNOWLEDGMENTS We gratefully acknowledge the engineering assistance of K. W. Doherty and the laboratory assistance of S. J. Molyneaux.
This work was supported by research grants DES7515017 and OCE75-21278 from the National Science Foundation.
LITERATURE CITED 1. Issacs, J. D. 1969. The nature of oceanic life. Sci. Am. 221:146-162. 2. Issacs, J. D., and R. A. Schwarzlose. 1975. Active animals 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 organic matter in the deep sea. Science 171:672-675. 4. Jannasch, H. W., and W. S. Maddux. 1967. A note on bacteriological sampling in sea water. J. Mar. Res. 17:185-189. 5. Jannasch, H. W., and C. 0. Wirsen. 1973. Deep-sea microorganisms: in situ response to nutrient enrichment. Science 180:641-643. 6. Jannasch, H. W., C. 0. Wirsen, and C. L. Winget. 1973. A bacteriological pressure-retaining deep-sea sampler and culture vessel. Deep-Sea Res. 20:661-664. 7. Morita, R. Y. 1972. Pressure 8.1 bacteria, fungi and blue-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. The growth and viability of sixty-three species of marine bacteria as influenced by hydrostatic pressure. J. Mar. Res. XI:10-18. 9. Paul, K. L., and R. Y. Morita. 1971. Effects of hydrostatic pressure and temperature on the uptake and respiration of amino acids by a facultatively psychrophilic marine bacterium. J. Bacteriol. 108:835-843. 10. Pope, D. H., and L. R. Berger. 1973. Inhibition of metabolism by hydrostatic pressure: what limits microbial growth? Arch. Mikrobiol. 93:367-370. 11. Schwarz, J. R., and R. R. Colwell. 1975. Heterotrophic activity of deep-sea sediment bacteria. Appl. Microbiol. 30:639-649. 12. Schwarz, J. R., A. A. Yayanos, and R. R. Colwell. 1976. Metabolic activities of the intestinal microflora of a deep-sea invertebrate. Appl. Environ. Microbiol. 31:46-48. 13. Seki, H., and D. G. Robinson. 1969. Effect of decompression on activity of microorganisms in sea water. Int. Rev. Gesamten Hydrobiol. 54:201-205. 14. Seki, H., E. Wada, I. Koike, and A. Hattori. 1974. Evidence of high organotrophic potentiality of bacteria in the deep ocean. Mar. Biol. 26:1-4. 15. Smith, K. L., Jr., and R. R. Hessler. 1974. Respiration of benthopelagic fishes: in situ measurements at 1230 meters. Science 184:72-73. 16. Wirsen, C. O., and H. W. Jannasch. 1974. Microbial transformations of some '4C-labeled substrates in coastal water and sediment. Microb. Ecol. 1:25-37.
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17. Wirsen, C. O., and H. W. Jannasch. 1975. Activity of marine psychrophilic bacteria at elevated hydrostatic pressures and low temperatures. Mar. Biol. 31:201208. 18. ZoBell, C. E. 1974. Some effects of high hydrostatic pressure on apparatus observed on the Danish Galathea deep-sea expedition. Deep-Sea Res. 2:24-32. 19. ZoBell, C. E. 1968. Bacterial life in the deep sea. Bull. Misaki Mar. Biol. Inst. Kyoto Univ. 12:77-96. 20. ZoBell, C. E. 1970. Pressure effects on morphology and life processes of bacteria, p. 85-130. In H. M. Zimmerman (ed.), High pressure effects on cellular processes.
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Academic Press Inc., New York. 21. ZoBell, C. E., and L. L. Hittle. 1969. Deep-sea pressure effects on starch hydrolyses by marine bacteria. J. Oceanogr. Soc. Japan 25:36-47. 22. ZoBell, C. E., and R. Y. Morita. 1957. Barophilic bacteria in some deep-sea sediments. J. Bacteriol. 73:563568. 23. ZoBell, C. E., and C. H. Oppenheimer. 1950. Some effects of hydrostatic pressure on the multiplication and morphology of marine bacteria. J. Bacteriol. 60:771-781.
Vol. 32, No. 3 Printed in U.S.A.
Undecompressed Microbial Populations from the Deep Sea' H. J. JANNASCH,* C. 0. WIRSEN, AND C. D. TAYLOR Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543 Biology, of Department
Received for publication 23 April 1976
Metabolic transformations of glutamate and Casamino Acids by natural microbial populations collected from deep waters (1,600 to 3,100 m) were studied in decompressed and undecompressed samples. Pressure-retaining sampling/incubation vessels and appropriate subsampling techniques permitted time course experiments. In all cases the metabolic activity in undecompressed samples was lower than it was when incubated at 1 atm. Surface water controls showed a reduced activity upon compression. The processes involving substrate incorporation into cell material were more pressure sensitive than was respiration. The low utilization of substrates, previously found by in situ incubations for up to 12 months, was confirmed and demonstrated to consist of an initial phase of activity, in the range of 5 to 60 times lower than the controls, followed by a stationary phase of virtually no substrate utilization. No barophilic growth response (higher rates at elevated pressure than at 1 atm) was recorded; all populations observed exhibited various degrees of barotolerance.
Extensive reviewing (7, 19, 20) of the literature on microbial activities at high hydrostatic pressures draws attention to the fact that all experimental studies in this area have been done either with surface-borne bacteria or microbial populations and isolates collected from the deep sea and held for some time at normal atmospheric pressure. ZoBell (19) characterized this problem of unavoidable decompression for laboratory studies: "Although many bacteria from the deep sea survived [the retrieval process], this observation fails to prove that some bacteria, possibly the most sensitive ones, were not destroyed by decompression. Answering this question may require the examination of deep sea bacteria at in situ pressures without subjecting them to decompression." In addition, one may be tempted to speculate that such high-pressure-adapted and decompression-sensitive bacteria, if they exist, would more likely be active under deep-sea conditions than would nonadapted forms. The problem of their recovery can be viewed in analogy to that of psychrophilic bacteria, which have a maximum growth temperature of about 20°C. Pure culture studies avoiding decompression will ultimately be necessary to prove the existence of decompressionsensitive bacteria and their possible barophilic behavior. Studies on the rapid decompression of natural populations of marine bacteria from a depth of 400 m only (13) showed some adverse effects I Contribution no. 3755 of the Woods Hole Oceanographic Institution.
due to "pressure shock." According to ZoBell (20), however, "most stock cultures are not injured by being compressed to 1000 atm within 1 or 2 minutes in nongaseous nutrient media and then immediately decompressed at about the same rate to 1 atm." The procedure of isolating bacteria from the deep sea implies, of course, decompression and a possible preselection of resistant organisms. Since the earlier work by ZoBell and Morita (22) in the Philippine Trench, it is known that substantial numbers of bacteria (most of them being psychrophilic) will grow on agar plates immediately after recovery from depths of more than 10,000 m. Recompression of these isolates to in situ pressure elicits different responses, which range from complete growth inhibition to various degrees of growth reduction. The following questions remain: (i) what is the in situ activity of those bacteria from the deep sea that can be isolated at 1 atm; (ii) are there bacteria in the deep sea that do not survive retrieval and decompression; and (iii) if so, do they differ in their in situ activity from those bacteria that do survive recovery? One way of attacking these questions is represented by in situ incubation experiments. By lowering sterile media into the deep sea for in situ inoculation as well as incubation, decompression is avoided. Control samples have to be collected for parallel incubation at normal atmospheric pressure and in situ temperature (2 to 4°C). Such studies have been carried out over several years and with large numbers of
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replicas. A variety of substrates (agar, starch, gelatin, and 14C-labeled acetate, mannitol, glutamate, and Casamino Acids) were deposited and recovered with the aid of the research submersible ALVIN on permanent bottom stations in the North Atlantic at a depth of 1,830 m (5) and in the Tongue of the Ocean at a depth of 1,960 m, with incubation periods of several weeks to 15 months. Surprisingly, the results have not been too different from those of analogous experiments done with surface-borne bacteria reported earlier (3), i.e., a similar retardation of growth and metabolism of the in situ incubated natural microbial populations, or pure and mixed cultures, as compared with controls incubated in the laboratory. These studies suffer from the distinct disadvantage, however, that rates must be determined from single end point measurements. Rates are estimated on the assumption that the activity determined is continuous and constant over the entire incubation period, a fact that cannot be proven. Furthermore, large numbers of replicate experiments necessary for statistical significance are difficult to obtain when depending upon diving opportunities with a submersible. We decided, therefore, to construct a bacteriological pressure-retaining sampler for the retrieval of deep-sea samples in the absence of decompression. To avoid transfer problems, the vessel is also used as a culture chamber. Any number of subsamples may be withdrawn during time course experiments without affecting the pressure within the vessel. The first instrument to be used to a depth of 2,000 m was built and tested in 1973. The detailed description of its operation (6) is principally the same as that for a more recent instrument that can be used for depths up to 6,000 m. The first results obtained with these instruments are described in the present report. MATERIALS AND METHODS Sampling. Two sampler/incubation vessels have been used for this study (Fig. 1), one for collecting water samples to depths of up to 2,000 m and the other for depths up to 6,000 m. Prior to sampling, two free-floating pistons (Fig. 2, a and b) are in their uppermost position, with sterile fresh water in the two sections of chamber B and a gas precharge in chamber C. When the sampler is lowered from the ship to the depth of sampling, the intake valve (f) in the upper end plate is opened by a messenger. As the sample enters the Teflon-coated chamber A, the two pistons move downward at a speed set by sterile fresh water in the upper section of chamber B, passing through a small orifice adjusted by the set-screw c to a filling time of 15 min. This pressure-snubbing device prevents the generation of high shear forces at the intake. Chamber C is precharged with nitro-
FIG. 1. Pressure-retaining sampler/incubation vessels. 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, 6,000 meters), Nitronic 50 stainless steel, trigger device attached. to assure a sufficiently large gas cushion at the final pressure. Precharge pressures are calculated on the basis of the estimated final depth of sampling and are, for example, 64 and 102 atm for the two samplers (Fig. 1) when operated at 2,000 and 6,000 m of depth respectively. The gas accumulation chamber is vital for preventing any substantial losses of hydrostatic pressure within chamber A due to small leakages or volume changes by expansion of the vessel. A check valve closes chamber A (maximum volume, 1 liter) when the filling is completed. The sample is well enough insulated to prevent temperature changes of more than a few degrees during retrieval through warmer surface water and transfer into a shipboard refrigerator set at 3°C. The actual minimum depth of sampling is determined by measuring the pressure within the sample. For incubation, the samplers are inverted and positioned over a magnetic stirrer (stirring bar d). In the larger sampler (Fig. 1), capable of withstanding 600 atm (safety margin of 4.0), the triggering level is affixed to the toggle valve located just prior to the snubbing orifice in the center section. For protection from contamination, the intake is covered by a cap containing a small sterile filter membrane. During lowering of the sampler, sterile water occupying the space between the intake and snubbing device is gradually compressed, causing some seawater to pass through the filter. The triggering mechanism uncovers the intake nozzle just before the filling starts. This precaution has been found necessary in earlier work using a tracer organism as an indicator of contamination from the unsterile surfaces of the sampling gear (4). Details gen
,
362
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JANNASCH, WIRSEN, AND TAYLOR en u: rrQ
ical Corp.; specific activities of 1.1 mCi/mg and 200 mCi/mmol, respectively) was added to unlabeled carrier solutions and introduced into the pressurized incubation vessels via the sterile transfer unit to a final concentration and activity of about 5 ,ug/ml wW and 0.005 ,Ci/ml, respectively. Due to the variable &\XX& degree of mixing during the introduction of a substrate sample, the initial concentrations are not identical from experiment to experiment and are individually indicated in the legends of the graphi!i~ z wIlllll,llfcally presented data (Fig. 3 through 5). Control collected in sterile Niskin samA4mlers at thesamples, same time and depth as the undecom,2 t///,' , pressed samples, were incubated in stoppered, 1liter Erlenmeyer flasks at in situ temperatures. The \ _ | \radiolabeled substrates were added prior to incuba_l tion. The closed gas phase in these 1-atm controls SA \ was kept as small as possible, never exceeding 10% of the sample volume, and was assumed not to affect the CO2 measurements. The same is held for the expected slight increase of the dissolved oxygen content. In the original samples oxygen was measured to be 290 to 300 ,uM or about 86 to 88% air saturation. The validity of the above assumptions was confirmed by a separate experiment (Fig. 5). Under the conditions of the experiments, oxygen limitation could not occur in the undecompressed samples. Complete oxidation of the available substrate to CO2 would result in a reduction of the r
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such as precharge calculations, design of intake cover, toggle valves, flow-snubbing orifice, etc. would go beyond the framework of this paper. The information is available upon request. A mixture of U-_4C-labeled amino acids or L-[U14C]glutamic acid (International Nuclear and Chem-
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DA YS FIG. 3. Incorporation (0) and respiration (0) of glutamate (initial concentrations, 5.58 and 5.78 pg/ ml) of water samples taken at depths of 1,800 and 3,000 m at the Bermuda transect station "MM" (34°45'N, 66°30'W) during incubation at 1 atm (initial concentration, 5.56 Mg/ml) and at in situ pressure and temperature (3.5°C).
UNDECOMPRESSED MICROBIAL POPULATIONS
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sample is decompressed through the appropriate port for analytical purposes. O_ o It should be noted that for a more efficient use of /° our two pressure vessels for incubation primarily, 0. .5 -------we recently built a special pressure-retaining sampling unit that concentrates a 3-liter sample during _______________________________-' 2. .5 to 15 ml by Nuclepore filtration. Concenfilling 0 N, trated and undecompressed subsamples can be 2.1o0 taken and stored in transfer units for later inocula1 atm tion into the prepressurized incubation vessels. By ° 1 .5 this means larger numbers of samples can be taken from various stations on a single cruise. The re1.1.0 _ /.-*10 C0°°-quired storage/transfer units are equipped with 313atm 0. 5 ____ small gas accumulators to prevent pressure loss. t v Data obtained from this sampling-storage-incuba^i==. t V tion procedure will be reported separately. 7 10 1 3 12 5 Analyses. Of each of the 12-ml subsamples taken DA YS after various intervals of incubation, 10 ml was filFIG. 4. Incorporation (a) and respiration (0) of tered through a 0.22-,um membrane filter (Millipore Casam,zino Acids (initial concentrations, 4.04 and 1.4 Corp.) and washed with 2 volumes of chilled seawapg/ml, of water samples taken at depths of1,700 and ter to determine the portion of substrate incorpo3,130 m at the North Atlantic station "DOS 2" rated into cell material and pools (incorporation). (38019>'N, 69041'W) during incubation at 1 atm (ini- Following the procedure of Wirsen and Jannasch tial concentration, 5.0 pg/ml) and in situ pressure (16), duplicate 0.3-ml samples were used for measurand temperature (3.5°C). ing 14CO2 production (respiration). Since remaining substrate and labeled dissolved intermediates were not measured, the term substrate utilization refers l otm, 4' latm, 22 to the total amount of substrate metabolized, i.e., the sum of incorporated and respired 14C-labeled F F_O-03 material. The radioactivity was counted using an Vm V / V 1< V Intertechnique SL-20 scintillation spectrometer. LL W was corrected for by the channels ratio *~-X Quenching f method. N,j A IS I. method. 1.
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FIG. 5. Incorporation (-) and respiration (0) of glutamate (initial concentration, 5.0 pg/ml) in surface seawater collected near Woods Hole during incubation at two pressures and two temperatures. The 1atm and 22°C experiment was conducted in the decompressed pressure sampler (@, 0) as well as in stoppered Erlenmeyer flasks (A, A).
concentration by no more than 50%. Subsampling. Subsamples (maximally 13 ml) can be removed from chamber A and equal portions of liquid medium can be introduced by attaching a transfer chamber (Fig. 1 and 2e). Sterile seawater or medium is contained in the transfer chamber and passed into chamber A by moving the hand-cranked piston. If a subsample is taken at the same time, no outside pressure source is needed for the operation. If a subsample of 10 ml is taken without the simultaneous addition of a liquid sample or compensation from an outside pressure source to chamber C, the pressure within the culture chamber will decrease about 3%. More details are given by Jannasch et al. (6). After detachment of the transfer unit, the suboxygen
The following data have been obtained on several separate cruises and geographical locations as indicated. The temperature, hydrostatic pressure, type of substrate used, and initial concentration of the substrate are indicated in the figures or their legends. Since the initial substrate concentrations vary (see above), the data on the degree of substrate utilization are given in
percentages.
In most experiments, the amount of substrate incorporated decreases after an initial peak due, probably, to the release of labeled material and products of autolysis. Identification of these materials is underway. For the estimation of rates and substrate utilization, only those data are used that were obtained during the period of increasing substrate incorporation. In the first experiment reported here (Fig. 3), water samples were taken at 1,800 and 3,000 m and incubated undecompressed as well as at 1 atm after the addition of glutamate. After 9 days of incubation at 180 atm, 3.5% of the initial amount of substrate was incorporated, as compared with a 34.5% utilization after 2 days at 1 atm. The corresponding values for respira-
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TABLE 1.. Metabolic ratios obtained from tion are 17.5% utilization after 9 days at 180 percentages of total substrate incorporated plus atm, as compared with 48.2% after 3 days at 1 atm. The estimated rate of substrate incorpora- respired (M, 100%o) over the percentage of substrate incorporated (I) by undecompressed and tion was 51 times and that of respiration 11 decompressed natural populations of marine times slower at in situ pressure than at 1 atm. microorganisms a Total utilization of glutamate after 9 days at 180 atm was about four times less than that at 1 Pressure (atm) Total substrate incorM/I porated(% atm. When the same experiment was done with 180 16 6.2 1 47 2.1 water samples taken from a depth of 3,000 m, 300 11 9.0 the percentage of substrate incorporated at 300 1 43 2.3 atm reached a plateau of 1.8% in 16 days, 313 29 3.4 whereas at 1 atm 28.5% was incorporated in 3 170 27 3.7 days. The corresponding values for respiration 1 44 2.3 were 18.5% at 300 atm in 16 days and 38% at 1 a Substrates: glutamate and Casamino Acids. atm in 3 days. Accordingly, the estimated rates for substrate incorporation and respiration Compare Fig. 3 and 4. were 64 and 11 times slower, respectively, at in situ pressure than at 1 atm. Total utilization of ratio was obtained at 313 atm than at 170 atm, glutamate in 16 days at 300 atm was about 3.3 but the values are still significantly higher than in the 1-atm control. times less than at 1 atm. Another experiment was conducted with CasThere were two more experiments conducted, amino Acids as the substrate (Fig. 4). Samples one in the Venezuelan Basin with samples colwere taken at depths of 1,700 and 3,130 m. At lected at 1,600 m and one in the North Atlantic 170 atm, the amount of Casamino Acid incorpo- with samples from 2,600 m. The results are rated was 9.4% after 4 days and the amount principally the same as those reported above, respired was 23% after 6 days. The correspond- but the data are somewhat less complete and ing data for 313 atm are 10.3% incorporation therefore not included. In most of these experiments, the incubation and 22% respiration after 6 days. A 1-atm control, which was done only for the sample taken was continued for as long as 5 weeks, with no at a depth of 3,130 m, showed a maximum significant changes in the measurements. Casamino Acid incorporation of 21% in 5 days When the vessels were brought to room temperand a maximum respiration of 47% in 8 days. ature and decompressed, incorporation as well The rates of substrate incorporation and respi- as respiration activities increased sharply after ration were 9 and 4.8 times slower, respec- a brief lag. To complete the picture of combined pressure tively, at 313 atm than that in the decompressed sample. Total utilization of Casamino and temperature effects, glutamate incorporaAcids after 6 days at 313 atm was about 2.1-fold tion and respiration of a surface seawater samless than that at 1 atm. ple (collected in December 1975 near Woods The effect of hydrostatic pressure on sub- Hole) were measured at 1 atm as well as at 190 strate incorporation relative to respiration may atm and at 4°C as well as at 22°C. The data be expressed by the ratio M/I: ratio of the total presented in Fig. 5 show the distinct effect of a amount of substrate incorporated plus that res- 190-atm increase in pressure, which was, howpired (i.e., metabolized [M]) to the total ever, less pronounced than the 18°C drop in amount incorporated (I), both expressed in mi- temperature. Pressure primarily reduced incorcrograms per milliliter. The data for these ra- poration, whereas a low temperature resulted tios were obtained over the period of initial in a pronounced lag. In combination, both efactive growth, i.e., prior to the decline of sub- fects are expressed when the sample was incustrate incorporation. Table 1 shows an increase bated at 190 atm and 4°C. in the M/I ratios with increasing pressure for In the samples incubated at 1 atm, the perthe data presented in Fig. 3 and 4. In compari- centage of the substrate utilized (about 95%) son to the 1-atm controls, increased pressure was unaffected by the 18°C temperature differresulted in a greater portion of total substrate ence. In the samples incubated at 190 atm, to be respired than incorporated. The agree- substrate utilization decreased from 85% meament between the 1-atm data is excellent. In sured at 220C to 74% at 40C. the experiment using Casamino Acids, a generIn a parallel experiment, incorporation and ally preferred substrate, a slightly lower M/I respiration at 1 atm and 220C were measured in
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1-liter Erlenmeyer flasks. These data (Fig. 5) were nearly identical with those obtained in the sampler/incubation chamber under the same conditions. These results rule out possible apparatus effects as being responsible for the low maximum utilization of substrate in pressurized cultures (Fig. 3 through 5). DISCUSSION In general, the data of this study confirm our earlier observations (5) on decreased microbial activities in the deep sea. Adding to this information, originally obtained by end point measurements after prolonged in situ incubation, the present time course experiments showed the rates of activity not to be constant. An initial incubation period of several days to several weeks, in which the activity was roughly 5 to 60 times slower than in the controls, was followed by a stationary phase, a virtual arrest of any further activity even though about 80% of the available substrate remained unutilized. It is apparent that increased hydrostatic pressure and decreased temperature resulted in both a retardation and a reduction of substrate utilization. Undecompressed cultures have been kept long beyond the initial phase of activity without observing any further change in the amounts of incorporated and respired labeled material. Continued metabolism at low levels of activity is likely although not detectable by our measurements. When, at the termination of the experiments, the pressure was released and the temperature was raised, the metabolic activity was found to increase or to "recover" after a brief lag. This response indicates the absence of an irreversible inhibitory effect. At this time, the limited utilization of substrate at increased pressure and low temperatures is unexplained and will be subject to continued research. This will include measurements of unutilized substrate remaining and determination of possible dissolved labeled intermediates. More specific points of discussion are the following. (i) Decompressed natural populations of microorganisms collected at depths in the range of 1,700 to 3,100 m showed a considerably increased metabolic activity relative to undecompressed populations. Vice versa, the metabolic activity of surface-originated populations was markedly reduced when incubated under pressures of the range indicated above. From these observations it appears that populations from deep water and those from surface waters behave similarly. It must be noted, however, that it is not possible to differentiate between
365
different components of the natural populations until pure cultures can be obtained. Furthermore, since there is no reliable way at this time to assess the number ofthe metabolically active cells in the natural population, we did not express our data on a "per cell" basis. Experimental work with undecompressed pure cultures appears to be necessary. (ii) The processes involving substrate incorporation were more pressure sensitive than was respiration, as reflected by the increased M/I ratio. This notion is reviewed in some detail by Pope and Berger (10). Data of Paul and Morita (9) showed that hydrostatic pressure and low temperatures affected glutamate incorporation more than did respiration in a psychrophilic marine bacterium. Schwarz and Colwell (11) recently reported that, of the total substrate consumed, respiration increased approximately 22% in pressurized samples of deep-sea sediment bacteria in comparison to 1-atm controls. These results were principally confirmed in our recent study (17) with a number of psychrophilic isolates. In some preliminary pure culture experiments, we have tried to include viable cell counts in order to express the data on a "per cell" basis. The results appear to indicate a higher amount of substrate metabolized per viable cell at elevated pressure than at 1 atm. Until such data can be verified by appropriate counting techniques, we ascribe this result to a decrease in viable cell numbers that has occurred during decompression. Obviously, viable counts from decompressed natural populations will be even less useful for expressing the M/I ratio on a per cell basis. (iii) If "barophilic" behavior is defined by higher rates of growth and metabolism at elevated pressure than at 1 atm, all responses of natural microbial populations observed in this study can only be described as "barotolerant"; i.e., the metabolic activities were reduced by the applied pressures to a variable degree. These results cannot be taken, of course, as proof for the absence of barophilic organisms that may escape detection within the gross reactions measured. Again, work with undecompressed pure culture isolates appears indispensable. The existence of truly barophilic microorganisms, in the above sense, was indicated in the early work of ZoBell and Morita (22) on a sulfate-reducing isolate from the deep sea. Seki et al. (14) found in one of two rubber bulbs (J.-Z. sampler) inoculated and incubated for 5 days at a depth of 5,200 m that bacterial growth on a peptone-yeast extract medium had occurred at
JANNASCH, WIRSEN, AND TAYLOR a higher rate than in a 1-atm control. No culture was kept. There is no direct proof and some doubt (18) that the rubber bulbs, due to a loss of elasticity at high pressure, actually fill at the same depth that they are triggered and opened. On the basis of our earlier results, we have speculated (5) that the principal site of microbial activity in the deep sea might be the intestinal tract of benthic animals, where prevailing high nutrient levels could support a population of pressure-adapted microorganisms. There are no measurements done yet on undecompressed gut samples. Our studies on decompressed gut samples from deep-sea fish, molluscs, arthropods, and echinoderms have resulted in no more than pronounced barotolerant responses, when studied under in situ pressure, amounting to a 10 to 90% reduction of the activity observed at 1 atm (unpublished data). Schwarz et al. (12), however, reported no or little effect of 750 atm on the increase of viable cell counts when incubating a diluted sample of gut content of amphipods recovered from 7,000 m. The CO2 production from starch was 2.9% higher at in situ pressure than at 1 atm, indicating a slight barophilic behavior. When these enrichments were decompressed and pure cultures were isolated, they showed typical barotolerant behavior, i.e., a reduction of activity in the range of 45 to 65% at 700 atm as compared with 1 atm, agreeing with other reports on barotolerant bacteria (8, 21, 23). Discussions of these and other data which indicate relatively low rates of microbial activity at high pressure, as compared with 1 atm, should not overlook the fact that in situ activities are still substantial in proportion to the amount of organic matter reaching the deep sea floor by sedimentation and do obviously not lead to any abnormal accumulation of organic materials. Artificially added organic waste materials, however, not readily available as food for the macrofauna, may decompose at a considerably slower rate in the deep sea than in shallow water. There is, at the present stage of this research, an interesting discrepancy in the data on the behavior of microorganisms in the deep sea versus observations made on benthic animals. Some of the latter have been photographically recorded to respond relatively quickly to submerged bait, locating and consuming it within periods from several hours to a few days (1, 2). Although the only in situ measurement of a metabolic process (oxygen consumption) to date on deep sea fish (15) resulted in relatively slow rates, R. Turner (personal communication) observed trapped specimens of benthic
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bivalves and arthropods in cages of small mesh size, implying a relatively high rate of growth within known time periods. Although still qualitative, such observations indicate a most intriguing variance to the strictly microbiological data. The apparent inconsistency might be resolved on the level of competition between microorganisms and higher forms of life under the particular conditions of food distribution in the deep sea. ACKNOWLEDGMENTS We gratefully acknowledge the engineering assistance of K. W. Doherty and the laboratory assistance of S. J. Molyneaux.
This work was supported by research grants DES7515017 and OCE75-21278 from the National Science Foundation.
LITERATURE CITED 1. Issacs, J. D. 1969. The nature of oceanic life. Sci. Am. 221:146-162. 2. Issacs, J. D., and R. A. Schwarzlose. 1975. Active animals 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 organic matter in the deep sea. Science 171:672-675. 4. Jannasch, H. W., and W. S. Maddux. 1967. A note on bacteriological sampling in sea water. J. Mar. Res. 17:185-189. 5. Jannasch, H. W., and C. 0. Wirsen. 1973. Deep-sea microorganisms: in situ response to nutrient enrichment. Science 180:641-643. 6. Jannasch, H. W., C. 0. Wirsen, and C. L. Winget. 1973. A bacteriological pressure-retaining deep-sea sampler and culture vessel. Deep-Sea Res. 20:661-664. 7. Morita, R. Y. 1972. Pressure 8.1 bacteria, fungi and blue-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. The growth and viability of sixty-three species of marine bacteria as influenced by hydrostatic pressure. J. Mar. Res. XI:10-18. 9. Paul, K. L., and R. Y. Morita. 1971. Effects of hydrostatic pressure and temperature on the uptake and respiration of amino acids by a facultatively psychrophilic marine bacterium. J. Bacteriol. 108:835-843. 10. Pope, D. H., and L. R. Berger. 1973. Inhibition of metabolism by hydrostatic pressure: what limits microbial growth? Arch. Mikrobiol. 93:367-370. 11. Schwarz, J. R., and R. R. Colwell. 1975. Heterotrophic activity of deep-sea sediment bacteria. Appl. Microbiol. 30:639-649. 12. Schwarz, J. R., A. A. Yayanos, and R. R. Colwell. 1976. Metabolic activities of the intestinal microflora of a deep-sea invertebrate. Appl. Environ. Microbiol. 31:46-48. 13. Seki, H., and D. G. Robinson. 1969. Effect of decompression on activity of microorganisms in sea water. Int. Rev. Gesamten Hydrobiol. 54:201-205. 14. Seki, H., E. Wada, I. Koike, and A. Hattori. 1974. Evidence of high organotrophic potentiality of bacteria in the deep ocean. Mar. Biol. 26:1-4. 15. Smith, K. L., Jr., and R. R. Hessler. 1974. Respiration of benthopelagic fishes: in situ measurements at 1230 meters. Science 184:72-73. 16. Wirsen, C. O., and H. W. Jannasch. 1974. Microbial transformations of some '4C-labeled substrates in coastal water and sediment. Microb. Ecol. 1:25-37.
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17. Wirsen, C. O., and H. W. Jannasch. 1975. Activity of marine psychrophilic bacteria at elevated hydrostatic pressures and low temperatures. Mar. Biol. 31:201208. 18. ZoBell, C. E. 1974. Some effects of high hydrostatic pressure on apparatus observed on the Danish Galathea deep-sea expedition. Deep-Sea Res. 2:24-32. 19. ZoBell, C. E. 1968. Bacterial life in the deep sea. Bull. Misaki Mar. Biol. Inst. Kyoto Univ. 12:77-96. 20. ZoBell, C. E. 1970. Pressure effects on morphology and life processes of bacteria, p. 85-130. In H. M. Zimmerman (ed.), High pressure effects on cellular processes.
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Academic Press Inc., New York. 21. ZoBell, C. E., and L. L. Hittle. 1969. Deep-sea pressure effects on starch hydrolyses by marine bacteria. J. Oceanogr. Soc. Japan 25:36-47. 22. ZoBell, C. E., and R. Y. Morita. 1957. Barophilic bacteria in some deep-sea sediments. J. Bacteriol. 73:563568. 23. ZoBell, C. E., and C. H. Oppenheimer. 1950. Some effects of hydrostatic pressure on the multiplication and morphology of marine bacteria. J. Bacteriol. 60:771-781.
