Bacteria in the cold deep-sea benthic boundary layer and sediment–water interface of the NE Atlantic

FEMS Microbiology Ecology 33 (2000) 89^99

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Bacteria in the cold deep-sea benthic boundary layer and sediment^water interface of the NE Atlantic Carol Turley * Plymouth Marine Laboratory, Citadel Hill, The Hoe, Plymouth PL1 2PB, UK Received 13 March 1999; received in revised form 26 May 2000 ; accepted 14 June 2000

Abstract This is a short review of the current understanding of the role of microorganisms in the biogeochemistry in the deep-sea benthic boundary layer (BBL) and sediment^water interface (SWI) of the NE Atlantic, the gaps in our knowledge and some suggestions of future directions. The BBL is the layer of water, often tens of meters thick, adjacent to the sea bed and with homogenous properties of temperature and salinity, which sometimes contains resuspended detrital particles. The SWI is the bioreactive interface between the water column and the upper 1 cm of sediment and can include a large layer of detrital material composed of aggregates that have sedimented from the upper mixed layer of the ocean. This material is biologically transformed, over a wide range of time scales, eventually forming the sedimentary record. To understand the microbial ecology of deep-sea bacteria, we need to appreciate the food supply in the upper ocean, its packaging, passage and transformation during the delivery to the sea bed, the seasonality of variability of the supply and the environmental conditions under which the deep-sea bacteria grow. We also need to put into a microbial context recent geochemical findings of vast reservoirs of intrinsically labile organic material sorped onto sediments. These may well become desorped, and once again available to microorganisms, during resuspension events caused by deep ocean currents. As biotechnologists apply their tools in the deep oceans in search of unique bacteria, an increasing knowledge and understanding of the natural processes undertaken and environmental conditions experienced by deep-sea bacteria will facilitate this exploitation. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Deep-sea; Decomposition ; Bacterium ; Sediment; Aggregate; Biotechnology

1. Introduction The deep-sea represents a signi¢cant long-term sink in the global carbon budget and can e¡ectively remove carbon for hundreds to millions of years. About 75% of the integrated bacterial biomass from surface waters to deepsea sediments is found in the top 10 cm of sediment [1]. The bacteria in oceanic and coastal sediments constitute around 76% (ca. 3.8U1030 ) of all global bacteria (ca. 5U1030 ) with around 13% (ca. 6.6U1029 ) of the total global fraction being found in the upper 10 cm of deep-sea sediments [2,3]. In the NE Atlantic deep-sea benthic boundary layer (BBL) and sediment^water interface (SWI), the temperatures are low (ca. 2³C at 4500 m), pressures high (450 atm

* Tel. : +44 (1752) 633292; E-mail : [email protected]

at 4500 m) and food is severely limiting. The deep-sea SWI can be viewed as representing the interface comprising both water and sediment between the £ux of materials carried through the water column and their incorporation within the sediment record. The BBL is the layer of water above the SWI with homogenous temperatures and salinity [4] which, at times, is enriched with resuspended detritus through increased bottom currents [5]. This detrital or particulate organic matter (POM) raining from the richer productive surface layer of the ocean often forms a seasonal £u¡y layer on the SWI and is the nutritional basis for life. For this reason, it is essential to understand the temporal and spatial processes occurring throughout the water column from POM production, through primary production in surface waters, to its aggregation and transformation as it sinks to the deep-sea bed. Respiration in the SWI is dominated by bacteria [6] and they play a major role in the decomposition of material on the deepsea bed [7,8] being able to consume at least 13^30% of the total biological consumption of organic carbon [9]. They

0168-6496 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 0 5 8 - 1

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can respond rapidly to the arrival of material, producing enzymes that break it down to smaller fractions, which they can incorporate to fuel their metabolism. This process dominates the biogeochemistry of the SWI and, therefore, the rate and nature of what gets laid down in the sediments. Residual biogenic material provides a sedimentary record of previous changes in ocean productivity, which can be linked to data on climate change and previous CO2 concentrations. Physical and biological conditions drive the fate and residence time of this material. Important physical factors include pressure, temperature, topography, boundary currents and advection while biological ones include degree and timing of £ux via pelagic^benthic coupling, remineralisation, bioturbation and bioirrigation. Anthropogenic input to the SWI will also be a¡ected by such forces. Bacteria, which contribute the greatest biomass, hydrolytic enzyme activity and rates of carbon turnover in this layer [1,9,10] and contribute to all the major biogeochemical processes (e.g. C, N, P, Mn, Fe, S, O cycling), will be in£uenced by many of the physical and biological forcings in the SWI and their response a¡ects the overall fate and residence time of this material in the marine environment. As work on the deep NE Atlantic is still relatively limited, extrapolation of results and processes found in other marine environments may be drawn upon to highlight areas in need of conceptual development as well as hands-on research.

ocean [7,22]. While 10^40% of primary production may leave the upper 100 m [20,23] of the NE Atlantic, most gets remineralised during its decent [20,24] so that only a few percent of the surface primary production arrives on the deep-sea bed [20] (Fig. 2). The carbon ¢xed by primary producers in the upper ocean is recycled to the atmosphere within weeks. However, the carbon aggregated into sinking particles e¡ectively removes the carbon for centuries (mid and deep waters) or millions of years (when laid down in sediments) (Fig. 2). The £ux of particles and their degradation during and after their decent is, therefore, of key importance to the global carbon cycle as well as to the delivery of food to deep-sea organisms. Particle £ux to the sea bed can be measured, and its seasonality determined, by sediment traps moored above the BBL (Fig. 1.4). The seasonality of events, from surface production to arrival of macroaggregates on the sea bed, has been captured in the sequence of time-lapse photographs of the sea £oor at 4025 m depth in the NE Atlantic (Fig. 1.5). Many taxa respond to this seasonal in£ux of material such that populations of opportunist species may increase and reproduction and growth cycles of some metazoans may be regulated [12]. Perhaps the greatest opportunists are bacteria re£ecting their rapid response by increased enzyme production, DNA and protein synthesis, respiration and on occasions an increase in sediment bacterial biomass after the seasonal in£ux of POM [1,20]. 3. Life on an aggregate

2. Food supply to the deep The supply of POM to the sea bed is the major determinant of abundance and activity of deep-sea benthic microbiota, meiofauna, macrofauna and deposit feeding megafauna [2,10^12]. This supply of particles originates from primary production in surface waters, such as that seen in the satellite image in Fig. 1.1, which, in the NE Atlantic, is mainly expressed in a phytoplankton bloom during the spring. The macroaggregates of phytodetritus (Fig. 1.2; commonly known as `marine snow') and faecal pellets, which are the major components of the £ux to the SWI, are generally produced in the upper 100 m of the water column [13], and also exhibit strong seasonal and diurnal variation [14]. They are comprised of a wide range of species and sizes of living and dead phytoplankton and zooplankton [15,16] held together by a sticky matrix of mucopolysaccharides [17] (Fig. 1.2) produced by phytoplankton cells or mucus feeding webs of zooplankton such as Appendicularia [18]. These sedimenting particles also contain an enriched and active population of bacteria (Fig. 1.3) relative to free-living bacteria [19^21] which are grazed by bacterivorous £agellates (Fig. 1.3). Detrital material recovered from the deep-sea bed is comprised of a similar mixture of both living and dead cells which characterised the organisms growing in the surface of the

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The aggregate microniche is recognised as an area of nutrient enrichment containing higher concentrations of both active and dead phototrophic and heterotrophic planktonic cells than in the surrounding seawater [15,19]. Bacteria clearly play an important role in the remineralisation and solubilisation of the particulate organic carbon (POC) such that many aggregates will be both formed in the warmer euphotic zone and be recycled there. However, many, most likely the larger, stronger ones, do escape to the midwaters where decomposition rates may be reduced by the cooler temperatures. Indeed, temperature variation experienced by organisms on an aggregate can be quite considerable. For example, temperature in the upper 20^ 40 m of the NE Atlantic during summer is often around 18³C. Below this, there is an area of rapid temperature change, the thermocline, where temperature falls to around 12³C and then decreases gradually so that temperatures at depths of around 1000, 2000 and 4500 m are around 5, 3 and 2³C, respectively. In addition, protein and DNA synthesis of bacteria attached to the aggregates in the surface waters may be drastically in£uenced by the high pressures (100 atm every 1000 m) as well as the low temperatures experienced during the sinking of large particles [25]. The reduced microbial activity on such particles may contribute to the delivery of relatively undegraded

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aggregates to the deep-sea bed. Molecular genetic techniques indicate that bacteria attached to oceanic macroaggregates may share few RNA types with free-living bacteria and that relatives of the attached phenotypes are often associated with surfaces and can degrade a wide range of polymeric compounds [26]. Attached bacteria also tend to be substantially larger and contain more DNA than free-living bacteria [27]. Upon arrival on the sea bed, the particulate organic £ux can support a fast growing population of deep-sea bacteria [7,8]. However, free-living bacterial populations change with depth [28] and it is possible that scavenging of freeliving bacteria, which may be adapted to life in the deepsea, occurs during the decent of the particles. In addition, phytodetrital enrichment experiments carried out under in situ pressure and temperature show that deep-sea bacteria associated with the SWI can respond to enrichment within hours or days [8,29] and contribute substantially to its decomposition. Turley and Lochte [8] found that 28% of detrital carbon was degraded in the ¢rst 23 days after decomposition started. The aggregates may also be resuspended into the BBL because of bottom currents and be redeposited [5]. Whether such events stimulate or inhibit microbial activity in the deep-sea remains unknown but laboratory experiments on shallow water aggregates have shown their continued suspension results in higher microbial production and respiration [30]. 4. Solubilisation of POM Bacteria produce hydrolytic enzymes prior to POM decomposition so that POM is cleaved into smaller molecules, which can support bacterial metabolism [31]. Studies on deep-sea sediment bacterial exoenzymes indicate that their production is regulated by the supply of substrates and nutrients, some enzymes being induced and some repressed, but in general POM induced higher enzyme production than dissolved organic matter (DOM) [32,33], suggesting that bacteria in the SWI respond directly to the seasonal fall of detritus. These enzymes and other measures of activity are highest in the upper 1 cm of the sediment [10] and in the overlying detrital or `£u¡' layer [34]. In continental slope sediments, glycosidase activity was positively correlated to the £ux of phytodetritus, while peptidase activity increased with water column depth and reduced food availability [35]. Boetius and Lochte [33] suggest that high peptidase activity may be indicative of oligotrophic (food limiting) conditions. Addition of organic nitrogen resulted in signi¢cant bacterial growth and indicated that nitrogen may be limiting growth of sediment bacteria [33]. Large additions of glucose provided enough energy for bacterial growth presumably using sedimentary nitrogen [33]. It therefore seems that the supply of organic nitrogen in sinking detritus will be an important control on bacterial growth while input of labile carbon

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may facilitate a greater decomposition of sedimentary organic compounds [33]. These observations demonstrate the importance of nutrient cycling (and therefore availability) in controlling the microbial response to changes in their environment, and the importance of enzyme production and regulation on the survival of bacteria in the deepsea SWI [31]. Rates of POM degradation and the e¤ciency at which this is converted into biomass depend largely on the proportion of labile material [1,8]. Under in situ pressure and temperature, conversion e¤ciency decreases from around 50% on relatively fresh material to around 10%, with increasing age of the material and there is evidence that deep-sea adapted bacteria are more e¡ective at degrading the less labile material than their upper water column counterparts [1,8]. It may not be totally surprising, therefore, that activity of an extracellular protease produced by a bacterium isolated from 6500 m was enhanced under high hydrostatic pressure [36], suggesting that the protease may be adapted to the high pressure found in the deepsea. The metabolic versatility of deep-sea bacteria may, therefore, enable the breakdown of compounds that are unavailable to other organisms [37]. Deming and Yager [2] compiled the few existing deep-sea sediment datasets and found a signi¢cant relationship between both bacterial biomass and bacterial dissolved organic carbon (DOC) utilisation and vertical POC £ux. Typically, 90% of [14 C]amino acids fed to sediment bacteria was respired over 2^5 days, leaving the remaining small amount for cell maintenance and growth. This can decrease to ca. 30% at high latitudes and may well be due to an increase in both quantity and quality of the sedimenting particles leaving more for maintenance and growth [2]. Indeed, some seasonal increase in benthic bacterial biomass was observed by Lochte [1] in the N Atlantic, where phytodetritus had also been observed and bacteria were seen to double rapidly [7]. This can be attributed to a response of the benthic bacteria to the seasonal vertical £ux of POM [20]. Stimulation of bacterial [3 H]leucine incorporation and enzymatic activity occurs in deep-sea sediment cores when enriched with detritus. Furthermore, Pfannkuche [6] compared sediment community oxygen consumption with bacterial growth and respiration in the NE Atlantic and found that 60^80% of the doubling in respiration between April (before detritus arrived) and July/August (after detritus arrived) is due to microorganisms inhabiting the SWI. In addition, a signi¢cant positive, exponential relationship between bacterial thymidine incorporation rates (a measure of bacterial production) and POC concentrations was found in the NE Atlantic SWI (upper 1 cm sediment and overlying detrital layer) [34]. When comparable data from the Soloman and Coral Seas were superimposed on this, the relationship held (P s 0.001). Should this relationship hold for other oceanic sediments, then prediction of deep-sea sediment bacterial production and their role in

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early sediment diagenesis may be possible from more simple measurements of sediment or detrital POC. All of these investigations indicate that the bacterial growth in the deep-sea of the NE Atlantic is food-limited. Hence, bacteria are implicated in a major way in decomposition in the deep-sea SWI. The large-scale consequences of this decomposition are produced by processes occurring at very small spatial and temporal scales. In addition, the delivery of labile material to the sea bed is

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not homogenous but rather packaged in the form of discrete aggregates and faecal pellets. The in£uence of such small-scale heterogenous processes and their impact on large-scale processes on the SWI has been shown to be important in the Norwegian^Greenland Sea [38] at depths of ca. 1750 m but received little attention in deeper waters. Since many of the enzymes produced by bacteria are attached to the bacterial membrane, bacteria must come in contact with POM to hydrolyse it. There is evidence that

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Fig. 1. 1: SeaWiFS surface colour image of the NE Atlantic on 10 May 1998 showing the development of the seasonal spring phytoplankton bloom o¡ Ireland. Chlorophyll is blue when concentrations are low, increasing through green and yellow to high concentrations at orange and red. Black is cloud cover. Images captured and archived by the NERC Satellite Receiving Station, Dundee, and processed by the Remote Sensing Group, Plymouth Marine Laboratory. Data courtesy of the NASA SeaWiFS Project and Orbital Science Corporation. 2: Epi£uorescent photomicrograph of a macroaggregate from the surface waters in the NE Atlantic, showing a range of red auto£uorescing (excitation at 450^490 nm) phytoplankton cells containing chlorophyll. Scale bar = 200 Wm. Taken by Dr C. Turley. 3: Epi£uorescent photomicrograph under blue excitation (450^490 nm) of dispersed detritus collected from the deep-sea bed at 4500 m in the NE Atlantic, stained with the DNA £uorochrome AO showing small orange rod-shaped bacteria and the larger, yellow £uorescing bacterivorous £agellates. Scale bar = 10 Wm. Taken by Dr C. Turley. 4: Time-series of sediment trap collecting cups. The cone shape of the trap funnels sedimenting particles into the cups which have been previously ¢lled with a preservative. The sequence of cups shown here containing settled material are from sediment traps deployed in the NE Atlantic in 1989 at 3200 and 4400 m water depth. The seasonality of sedimentation is clearly visible and is due to the £ux of material from the increased productivity during the spring bloom in the surface waters about 4 weeks previously. Courtesy of Dr P. Williamson, University of East Anglia. 5: `Bathysnap' time-lapse photographs of the sea bed at 4025 m depth in the NE Atlantic between 1 May and 10 August 1983. Initially, there is a progressive build-up of detrital material covering the sea bed, visible as dark patches overlying the lighter sediment. After 14 July, there is a progressive decrease. The mound in the centre is 18 cm across. Courtesy of Dr R. Lampitt, Southampton Oceanography Centre. 6: The Dunsta¡nage Marine Laboratory (DML) benthic lander (made by KC Denmark), which has the capability of sampling microscale variations in pore-water chemistry in pro¢le mode or of determining benthic £uxes when ¢tted with a box core benthic chamber. Oxygen electrodes in the chamber measure benthic community respiration. Syringes can sample water in the chamber to enable estimates of chemical benthic £uxes (e.g. DOC and inorganic nutrients). It can be deployed on the sea bed for several days or weeks and released from the ballast weight by an acoustically operated mechanism. A hydraulically operated shovel captures and retains the sediment for shipboard analysis and buoyancy spheres at the top carry the entire mooring to the surface after activation of the release. The lander is 4 m high. Courtesy of Dr K. Black, DML. 7: A mega corer (manufactured by Bowers and Connelly, Oban, a hydraulically damped multiple corer) during retrieval with eight, 10 cm internal diameter, plastic core tubes containing relatively undisturbed deep-sea sediment cores and overlying water. The photograph was taken during May 1998 in the NE Atlantic during a BENBO cruise during the surface water spring bloom (1) prior to the sedimentation of detritus to the sea bed. Taken by Dr C. Turley.

6

deep-sea bacteria from the SWI colonise aggregates prior to decomposition [8]. It is likely that contact with the bacteria-rich sediments and movement of the phytodetrital layer may result in faster incorporation of bacteria into the detritus. High sediment enzymatic activity has been associated with foraminifera in the deep waters (ca. 1750 m) of the Norwegian^Greenland Sea and it has been proposed that these Protozoa may contribute signi¢cantly to the total pool of hydrolytic enzymes [38], increasing DOM concentrations and stimulating bacterial growth. However, on incubating agglutinated foraminifera from the NE Atlantic in ¢ltered seawater, I have observed no increase in enzymatic activity until after the organisms died and decomposition commenced. An alternative explanation to the observations by Meyer-Reil and Ko«ster [38] may be that both bacteria and foraminifera had responded to an organic input by increasing their numbers and/or activity [12] or that the foraminifera are grazing the dense populations of bacteria [39]. Such conjectures demand further work on the deep-sea microbial food web and trophodynamics. 5. The sediment reservoir, resuspension and desorption Deep-sea sediments contain a vast reservoir of organic carbon and are a possible source of the old (4^6000 years) refractory DOC found in deep water [40]. There are substantial gradients of DOC and dissolved inorganic carbon (DIC) across the SWI implying their di¡usion from the sediment to the water column [41]. However, compared to the overlying water, the DOC in the sediment is greatly enriched in 14 C, indicating that the DOC supplied by the sediments to the water must be relatively young and that

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its remnant ages in the water column itself [41]. The upper (6 1 cm) sediment microlayer at the SWI is known to harbour the highest bacterial productivity and highest concentration of bacteria and their enzymes [10,38, 42,43], so this is the likely region of intensive microbiological processing of the DOC di¡using from the underlying sediment. This would also explain the DIC gradients across this interface through intensive conversion of DOC to DIC via bacterial respiration. A recent study has revealed that a large proportion of organic matter (OM) in marine sediments from ca. 650 m water depth, is intrinsically labile material stabilised through sorption onto mineral matrices [44]. This, presumably, makes it inaccessible to bacterial enzymatic hydrolysis. Once desorped, s 70% of the OM, having been present in the sediment for up to 500 years, was remineralised by bacteria within 6 days [44]. Such desorption may also occur in the deep-sea through increased particle^solute interaction during resuspension events caused by bottom currents and storms in the BBL [5]. This could make OM available for bacterial utilisation in the BBL as well as in the SWI once deposition reoccurs. Such desorption events have not yet, to my knowledge, been studied in deep-sea sediments but, should they occur, may result in organic enrichment. This may be particularly important during periods when there is little or no £ux of material from surface waters and, therefore, desorped OM may act as a food supply to bacteria in the SWI in otherwise extensive `lean' periods. The question of survival mechanisms of bacteria during these potentially long periods, perhaps through dormancy and shifts in metabolism, remains unresolved [45]. Tantilisingly, pressure-dependent membrane proteins and lipids have been found in isolated barophiles from the deep-sea, which may enable the cells

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to have greater substrate a¤nity when nutrient concentrations decrease [45^48]. The hydrodynamics of the BBL may therefore a¡ect the rates of OM decomposition through mixing, resuspension [49] and desorption of organic inputs, both of which may in£uence colonisation of the particles and incorporation of particles into the sediment. For example, resuspension events in the NE Atlantic were measured by sediment traps moored in the BBL at 4465 m, 90 m above the sea bed, and were found to comprise of recently deposited resuspended material and geological sediment [5]. This was greatly enhanced during winter and was related to near-bottom currents [5]. There is evidence that this deeper trap received bacteria from resuspended sediments and colonised macroaggregates and that there may be enhanced growth of these deep-sea bacteria [24]. 6. Adaptation to the cold, deep ocean The low temperature and elevated hydrostatic pressure found in the deep NE Atlantic are only extreme to those organisms that have not evolved there over time, e.g. those carried there on particles and currents. The natural residents of this extensive environment are so well adapted that many are well suited to growth at high pressure and low temperature, many of these are barophilic (pressureloving), some of which can be obligate barophiles [50]. Barophilic bacteria have been found to play a predominant role in the turnover of radiolabelled glutamic acid in sediment box core samples [51]. However, for bacteria from sediment trap samples from above the BBL, highest turnover rates were found at surface pressures implying that barophilic bacteria do not contribute substantially to decomposition in the deep-sea until after the particle £ux arrives on the sea bed. Lochte and Turley [7] found that bacterial growth rates were similar at in situ and surface pressures on naturally occurring phytodetritus recovered from the deep-sea bed. This implies that both deepsea adapted and surface-originating bacteria may play a role in decomposition after the particle has arrived on the deep-sea bed. Up to 3U109 cells m31 day31 can be carried to the deep-sea bed on sedimenting particles in this region [24]. What remains unquanti¢ed is the degree to which the bacteria originating from surface waters on the particles are able to contribute to decomposition in the SWI and whether genetic exchange with indigenous deep-sea bacteria can occur [24,45]. Pressure inducible genes which pressure-acclimatise bacteria experiencing such large vertical changes have been proposed [52]. Experimental work on cultures implies that there may be a tendency for increased barophily and oligotrophy with depth (synonymous with increasing pressure and decreasing food supply) [11]. However, growth rates of deep-sea bacterial assemblages under non-limiting nutrient conditions at in situ pressure and temperatures are similar to those of shallow water assem-

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blages [7,8,53], indicating that food resources are an important factor controlling growth. Free-living bacterial concentrations in the BBL (ca. 0.3^ 1.6U105 ml31 seawater) are several fold lower than those in the SWI (1^5U109 ml31 sediment) [42]. Rates of bacterial thymidine incorporation in the BBL and SWI also re£ect these sorts of di¡erence [54]. Bacteria in the SWI are, in general, larger (some s 10 Wm long) than those in the BBL with a greater frequency of dividing cells (per observation). Detrital enrichment experiments, carried out on free-living bacteria in the NE Atlantic BBL from 4500 m, under in situ pressure and temperature, showed that their mean cell volume increased from 0.065 Wm3 to 0.231 Wm3 in 5.5 days [8] with colonisation of the detritus very obvious. It, therefore, seems that small cell size of free-living bacteria in the BBL may be a response to starvation and, at least, some of the population were able to respond rapidly when food in the form of detritus became available. It may be a valuable exercise to relate mean cell volume to oligotrophy in the deep NE Atlantic and other oceans but I am unaware of such a study. The e¡ects of size reduction on cell viability and the e¡ects of other strategies for surviving starvation, such as attachment, are important issues in this extremely oligotrophic environment [45]. Barophilic bacterial populations also exist in the hindguts of holothurians (sea cucumbers), which feed opportunistically on phytodetritus and sur¢cal sediments. These bacteria may convert refractory organic compounds, not absorbed in the animal's foregut, into molecules that can be easily taken up by the host [55]. A range of di¡erent bacterivorous micro£agellates with a range of pressure tolerances have been isolated from sinking particles in the NE Atlantic [56]. Even at a depth of 4500 m, a barophilic £agellate of the genus Bodo has been isolated [42]. It grazed bacteria growing on phytodetritus and had a growth rate of 0.3 day31 under 450 atm and 2³C. The presence and rapid growth of these £agellates indicate that the microbial loop may well exist in the deep-sea. Bacteria may also be an important food resource for another important group of deep-sea organisms, the foraminifera [12]. 7. Sampling the deep-sea and methods application One reason the data are sparse in the deep-sea is the problem of sampling and maintaining the samples under in situ conditions. Pressure retaining samplers for retrieving undecompressed water have been designed and operated successfully [57]. However, retrieving sediment samples in a similar fashion is a more di¤cult problem currently requiring the use of manned submersibles or benthic `landers' (Fig. 1.6) resulting in few, but expensive data. A `lander' is an autonomous, unmanned vehicle that free-falls to the sea bed and then works independently on

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Fig. 2. A schematic diagram showing the £ux of carbon through the NE Atlantic water column to the deep-sea sediments. Amended from Dr P. Williamson [66].

the sea £oor [58]. More usually, researchers have worked on sediments retrieved using samplers (such as the corer seen in Fig. 1.7) where the sample gradually decompresses during retrieval and once on board ship, are recompressed in pressure vessels with a wide range of substrates added (see below) to investigate microbial metabolic rate processes. The e¡ects of decompression during retrieval have received little attention although laboratory cultures seem to be able to withstand brief periods of compression [57]. However, Bianchi and Garcin [59] have found a decrease in metabolic activity of deep-sea bacteria within the strati¢ed deep and warm (13³C) waters of the Mediterranean.

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What seems to be most critical is maintaining the very low temperatures characteristic of the majority of the deep-sea (ca. 2^5³C) [37,57,60]. Once the samplers are on deck, the samples need to be immediately removed to a constant temperature laboratory running at in situ temperature. In addition, all sample manipulation and incubation must be carried out at in situ temperatures prior to incubation under in situ pressure and temperature. All these requirements make the logistics and working conditions of deep-sea microbiology at the very least challenging. Bacteria in the deep-sea SWI are important in the transformation of organic material but due to their high respiration and low growth e¤ciency are often [53], but not

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always [1,20], non-growing. Thus there is often, but not always [10], a constancy of bacterial abundance [61] and following only incremental increase in bacterial biomass is not always an e¡ective method of determining their biogeochemical impact and can be misleading. That is, they may be expending the majority of their energy in enzyme production, respiration and cell maintenance rather than increasing their cell numbers. It is essential therefore to study a range of rate processes to get a more realistic understanding of the role of bacteria in biogeochemical transformations. Each method has limitations and assumptions associated with them [2,33,62] and measures a di¡erent aspect of microbial growth but their combined use may give a greater understanding of the microbial contribution to OM transformation in the SWI. Bacterial numbers and biomass can be determined by 4P6-diamidino-2-phenylindole and Acridine Orange (AO)staining epi£uorescent microscopy but a sonication and detergent step needs to be used to free bacteria from particles [24,63]. Microbial growth rates can be estimated by increase in bacterial counts [7] (but see caution above), bacterial respiration and incorporation rates of dissolved material by the [14 C]amino acid method [51,64] and remineralisation of model particulate material by 14 C-labelled algal cells [1]. The relative contribution of bacteria to total community respiration can be estimated from total community oxygen consumption using a benthic lander (Fig. 1.6) and bacterial respiration. Bacterial DNA and protein synthesis in seawater and detritus has been determined for some years by the addition of high speci¢c activity [3 H]thymidine and [3 H]leucine, respectively. Application of these two radioisotope techniques in seawater is now widely accepted. However, in sediments they are the best available methods although there are many unresolved problems in sediments [62]. These include potential non-incorporation by subsets of the sediment population, use of conversion factors from incorporation to production, e¡ects of slurrying the sediment, adsorption of the label to the sediments, extraction e¤ciency of the radiolabel and unmeasurable isotope dilution. However, there has been a recent attempt to resolve some of these problems in deep-sea calcium carbonate sediments [54]. To determine how bacteria achieve hydrolysis of the POM, the hydrolysis of a range of model £uorogenic substrates [32,65] can be measured. For example, 4-methylcoumarinyl-7-amide-labelled leucine hydrolysis, an analogue for measuring protease activity, will be an indicator of extracellular enzyme activity of relatively labile material. In contrast, methylumbelliferone (MUF)-labelled glucosaminide hydrolysis, an analogue for chitinaselike activity, will indicate enzymatic hydrolysis of more refractory substrates. Similarly, MUF-labelled K- and L-glucoside may be useful model substrates for carbohydrates which may be important components of macroaggregates arriving intact on the deep-sea bed. The ¢ndings

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of Boetius and Lochte [32,33] using these methods indicate that peptidase activity may be a useful indicator of food supply. The relative fraction of active, dormant and dead cells, as with other habitats, remains unquanti¢ed in the BBL and SWI but an important question. It is likely that, consistent with other aquatic and terrestrial environments, only a minor fraction of deep-sea bacteria have been obtained in culture and these may not be representative. The relative sparcity of such studies in the cold deep-sea, sampling di¤culties and the diversity of habitat found there may make culture of a larger proportion and representative species an even greater challenge than in other habitats. There are already many examples of active bacteria from the cold deep-sea that remain uncultured [45]. 8. Outlook In my mind there are two important areas for future research in the cold deep-sea. Firstly, we need to understand more fully the role of microorganisms in the cycling of OM and early diagenesis. This understanding is required on both small and large temporal and spatial scales. For instance, we need to determine the importance of microbial processes in the SWI and within aggregates or animal burrows and the di¡erences in rates of microbial processes between oceans and ocean regions of di¡erent depth and degree of oligotrophy. We need to try to achieve this understanding in an unobtrusive way introducing the minimal artifacts through sampling. In addition, the role of environmental conditions (e.g. seasonal £uxes, deep-sea storms and resuspension events and interannual variations) needs to be incorporated in our understanding and conceptual and mathematical models of the microbial role in the biogeochemistry of the deep-sea and hence the cycling of carbon on a global scale [66]. Secondly, the deep oceans, the largest and perhaps earliest biosphere on Earth, are an enormous reservoir of bacteria and a source of unique microorganisms that may have evolved some 3.5 billion years ago. As yet this wealth is largely unexplored and certainly untapped. Perhaps one of the most tantalising ideas is that through the molecular study of deep-sea bacteria we may get an important insight into the origin of life and its evolution. For instance, a high pressure regulated system for gene expression has been found not only in deep-sea adapted bacteria but also in bacteria, such as Escherichia coli, adapted to growth at atmospheric pressure and foreign to the deep oceans. One suggestion to explain this is that the systems developed in a high pressure environment and may be conserved in organisms adapted to atmospheric pressure and may indicate that life emerged from the deep-sea [36,67], the earliest inhabitants being fueled by hydrothermal vents [68,69]. However, the future may lie in combining our new

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C. Turley / FEMS Microbiology Ecology 33 (2000) 89^99

changed perceptions of the deep-sea environment, consisting of a myriad of micro-habitats containing vast numbers of bacteria capable of a wide range of biogeochemical activities under `extreme' conditions, with our new found biotechnological tools so that we will meet the challenge of exploring more fully the oceans s 2000 m deep which, after all, comprise about 60% of the Earth's surface. Already, the application of molecular techniques to the deep-sea has resulted in huge advances in our understanding of phylogenetic diversity. For example, one recent and remarkable discovery, using rRNA sequences of marine microbial diversity, is the importance of widespread occurrence of Archaea in the World's oceans [70]. These prokaryotes have been found to be important members of bacterioplankton communities in the deep ocean, in cold pelagic waters, in hydrothermal vents and in guts of deepsea deposit feeders [71,72]. The cold deep-sea has enormous biodiversity with estimates around 5^10 million species [73]. A case for high bacterial biodiversity in deep-sea sediments due to wide ranges in temperature, pressure and food resources has also been proposed [45]. With such potential microbial phylogenetic diversity, it is not surprising then that more countries are investing substantially in both the collection and culture of bacteria from the deep oceans to explore and exploit their metabolic diversity through biotechnological applications [45,74] by, for example, the development of high pressure or low temperature bioreactors, degradation of organic solvents and for producing steroids [36,75].

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Acknowledgements This is publication Number 4 of the Thematic Research Programme BENBO, carried out under award GST/02/ 1761 from the UK Natural Environment Research Council. My thanks to those who supplied the images and to J. Shackleford for compiling them and for typing the manuscript.

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