ContinentalShelfResearch,Vol.4, No. 4, pp. 445 to 464, 1985. Printedin GreatBritain. 0278-4343/85$3.00+ 0.00 O 1985PergamonPressLtd. Bacterial g r o w t h a n d the d e c o m p o s i t i o n o f particulate o r g a n i c c a r b o n c o l l e c t e d in s e d i m e n t traps HUGH W . DUCKLOW,*~" SUZANNE M . H I L L * t a n d WILFORD D . GARDNER* (Received 25 January 1984; in revisedform 13 July 1984; accepted 23 July 1984) Abstract--We have studied bacterial abundance and production in samples from sediment traps deployed for 1 and 100 days in several areas of the shelf and slope regions of the Middle Atlantic Bight, U.S.A. By making a series of assumptions about bacterial growth at the expense of POC in traps, we have estimated that the turnover time of organic particles collected in traps during long deployments is slow (mean 1500 4- 300 days), if only bacterial activity is consiaered. However the abundance and biomass of bacteria in traps is very high, ranging from 3 to 30 x 1011 cells gC -t , Le., 0.3 to 3% of the POC is bacterial carbon. Fifteen to 88% of the particles in traps were colonized by bacteria, but usually about half the particles had only 0 to 1 cell attached. Growth of bacteria was observed at all scales relevant to these trap deployments; over periods ranging from hours to weeks, at rates of 0.01 to 0.3 d -l . In spite of slow growth, bacteria appeared to be physiologically active in that [3 H]adenine and [3 H]thymidine were incorporated more rapidly into RNA and protein than into DNA. Total incorporation rates were high. We conclude that even relatively old (ca. I y) POC in sediment traps supports high levels of active bacterial biomass, but that POC decomposition is slow, so that bacteria may not be the principal agents of POC turnover following collection. INTRODUCTION SEDIMENTINGand resuspended particulate matter in the marine environment consists of a diverse collection of'large' particles including phytoplankton cells, zooplankton fecal pellets and molts, amorphous fecal matter, organic aggregates, marine snow, and mineral matter (BISHOP et al., 1977; Rowe and GARDNER, 1979; SHANKSand TRENT, 1980; FELLOWSet al., 1981). Any of these particle types may be falling through the water column either for the first time (primary particles), ingested and repackaged as new fecal material, or resuspended after deposition on the sea floor. Because of their rapid settling rates, many of these particles are not collected efficiently in Niskin bottles (SHEI.DONet aL, 1972; SASAKIand NISHIZAWA, 1981), and, even if collected, they often escape being sampled (GARDr~R, 1977). In the past few years, sediment traps have been used increasingly to sample large particles in the ocean and to estimate the rates of vertical particulate flux (REYNOLDSet al., 1980). But sediment traps are oases of rich accumulations of particulate organic matter and elevated biological activity in the relatively impoverished water column, and the extent to which biological processes alter trapped particles is poorly understood (GARDNERet aL, 1983). Most of the aforementioned particle types can be colonized by bacteria and could support bacterial activity. Bacteria are potentially major agents of mineralization of particulate *Lamont-Doherty Geological Observatory of Columbia University, Palisades, NY 10964, U.S.A. ~"Present address: Horn Point Environmental Laboratory, Box 775, Cambridge, MD 21613, U.S.A. 445 446 H . W . DUCKLOWet al. carbon and nitrogen (POC, PON), but their importance as mineralizers relative to protozoans, micro- and macrozooplankton is still not established (JOHANNES, 1965). Given the diversity of particle types, sizes, ages, and compositions, we expect a corresponding complexity of bacterial roles in decomposing particles. Investigations of bacterial relationships with sedimenting particles have only recently been undertaken using modern techniques (ITURRIAGA, 1979; DUCKLOWet a/., 1982; KARLand KNAUEg, 1984). Basic data on the bacteriology of materials collected in sediment traps is required for two reasons: to understand the fate of POC/N in traps, and to clarify the role of bacteria in decomposing particles in traps. Such data may also be useful for estimating the rates at which particles decompose as they settle through the water column. In this paper we report on patterns of colonization and growth of bacteria on particles and trap surfaces from a series of trap deployments in the Middle Atlantic Bight (MAB). In particular, we will present data on bacterial abundance changes and nucleic acid synthesis in materials from floating sediment traps deployed for 24 h and moored traps deployed for 3 to 6 months. It is not the purpose of this report to relate bacteriological parameters of the different collections to ambient conditions at each site. Rather we wish to demonstrate some of the variability in bacterial abundance and activity in these sediment trap collections in order to suggest preliminary answers to the questions: does sedimenting particulate matter support bacterial growth? and, what is the role of bacteria in the turnover of this material? It is difficult if not impossible to observe the bacterial role in decomposing sedimenting POC directly. Our approach as described below will he to measure the abundance and apparent growth rates of bacteria associated with the trap contents, and then use these results in a simple model of POC decomposition to estimate POC turnover times due to bacterial activity. By comparing these extrapolated results with observations of total POC turnover times, we hope to learn about the role of bacteria and also how to construct better models in the future. M A T E R I A L S AND METHODS All experiments and measucei~ents were made using samples of materials recovered from moored and floating sediment traps deployed in the Middle A t l a n t i c B ~ t during I980 to 1982 (Table 1). The floating s e d ~ n t traps and M4 moored traps ~able 1) were PVC cylinders similar to those described by ROWEand GARDNER(1979), The other moored traps (M 1 to M3, Table 1) were butyrate cylinders 7.6 × 38 cm with 1 cm wide by 5 cm deep baffles in the top. The samples from floating trap deployments were processed aboard R.V. Knorr within 2 to 4 h of recovery, as described more fully in DucgLow et al. (1982). Samples recovered at sea from moored traps were returned on ice to the laboratory at Lamont in plastic jars and stored in the dark at 5 °C, until further processi~ or subsampling. Subsamples from all traps were preserved in cell-free formalin immediately following recovery (floating traps and M4) or immediately following the arrival onice at Lamont (M1 to M3), for subsequent enumeration of pa~cles and bacteria b y e p l f l u o r e ~ m i c r o s c o p y and acridine-orange direct counting (AODC; HonmEet al., 1977). Determinations of total particulate matter in the traps were made by splitting, drying and weighing of the trap contents as described in ROWEand GARDNER(I979). POC determinations were made using Perkin-Elmer Model 120B CHN analyzers at Brookhaven National Laboratories (floating traps) a n d a t Lamont (M1 to M3). Particles in the traps were enumerated by egifluorescence microscopy and acridine-orange (AO)stained samples. We 447 Bacterial growth on POC in sediment traps Table 1. Details of sediment trap samples used in this study Sample Recovery date Deployment time (days) Latitude °N Longitude °W Bottom depth Trap depth Temperature at trap(s) (m) (m) (°C) i FI 3/10/81 F2 3/11/81 F3 3/17/81 / / 1 1 1 40020.7 ' 73053.6 ' 40 ° 14.8' 73 °55.5' 40 ° 19.8' 25 10 3 20 10 3 25 10 3 112 100 112 92 112 82 108 96 108 88 108 88 1005 655 5 1290 1075 5 73053.4 ' M la 10/2/81 111 M lb 10/2/81 111 M lc 10/2/81 111 M2a 10/2/81 112 M2b 10/2/81 112 M2c 10/2/81 112 M3 9/28/81 166 M4 11/5/80 99 38 o 10.0' 73054.7 ' 38 ° 10.0' 73054.7 ' 38 ° 10.0' 73054.7 ' 38° 18.1' 73046.3 ' 38 ° 18.1' 73 °46.3' 38 ° 18.1' 73046.3 ' 38005.2 ' 73047.2 ' 38°59.1 ' 72031.5 ' 7-9 (deployment) 12-13 (recovery) 6-9 (deployment) 11-12 (recovery) enumerated any particles which stained translucent or opaque green, red, or orange with AO, but counted bacteria separately. Bacteria were distinguished from particles by morphology and intensity of staining. Thus 'particles' as discussed below included mineral particles, amorphous organic matter, fecal pellets, phytoplankton cells and animal remains (molts, carcasses, etc.) which were visible in the stained samples, but not bacteria. Attached and free-living bacteria were enumerated separately. Bacteria were counted as 'attached' if they were seen in close contact with a stained 'particle'. Cells near, but not obviously in contact with visible particles were counted as free cells (KmcH~AN and MITCHELL, 1982; DUCKLOWetal., 1982). Free-living cells enter the trap when it fills following deployment, and by detachment from particles, or perhaps as a result of particle solubilization. We distinguish between free and attached cells in order to distinguish their respective importance in decomposing POC, using the model presented below. Several traps from deployment M3 (Table 1) were sampled in such a way that bacteria in the bulk contents and the bacteria colonizing the trap walls could be enumerated separately. Traps sat on deck for 0.5 to 1 h following recovery to allow particles to settle. The supernatant water was discarded and the samples were drained into plastic jars. The mucus-like material fouling the walls of the trap was gently scraped into a second jar. A control trap (M3) was deployed upside down to prevent particle collection; only the wall growth from this trap was collected. In these samples, free and attached bacteria were categorized as described above, even though free bacteria were contained in the mucus matrix. The contents of the six traps from deployments M 1 to M2 were incubated without shaking in plastic jars in the dark at 5°C following recovery and return to Lamont. This procedure 448 H.W. DUCKLOWet al. simulated storage in traps in situ. Subsamples from each jar were removed at intervals of I to 4 weeks following recovery for bacterial enumeration. The contents of traps from M4 were also stored for six months, then processed as described below. Besides following changes in bacterial abundance over time intervals ranging from hours to months in incubated samples, we also estimated bacterial activity from the rates of incorporation of the nucleic acid precursors [methyl-3H]thymidine and [2.3 H]adenine into DNA and RNA in short-term assays. The procedures described in FUI-mMAN and AZAM (1980) and in KARL et al. (1981) were followed exactly for [3H]thymidine or [3H] adenine incorporation, respectively. The specific incorporation of each precursor into DNA, RNA, and residue (protein) was determined by the sequential extraction procedure of KARL (1982). To examine the coupling between cell division and the incorporation of labelled nucleic acid precursors into macromolecules, the following experiment was performed on samples F2 and M4. The experiment with sample F2 was carried out at the in situ temperature (3°C) immediately following recovery of the sample. The experiment with sample M4 was performed at 15°C, following six months of storage at 5°C, in order to test the potential of trap contents to support bacterial activity after prolonged storage under in situ conditions. Samples were diluted by 1:3 (F2) or 1:4 (M4) with bacteria-free water from the same site, and then incubated with gentle shaking for 60 h (F2) and 10 h (M4). Periodically samples were withdrawn for bacterial counts, and for short-term (30 min) precursor incorporation assays. This experiment can be used to estimate growth rates o f the precursor-utilizing bacteria in the incubated samples (KmCHMANet aL, 1982), though we have not done so in the present paper. The radioactivity in all samples was assayed in 7 ml glass mini LSC vials with Aquasol II (New England Nuclear) on a Beckman LSI00 liquid scintillation counter. Quenching was estimated by the external standard ratio using a quenched [3 H]toluene standard series in glass minivials. D a t a analysis All data reported are means of replicate determinations +S.E., unless noted otherwise. Some of the data on the floating sediment trap deployments were presented in DOCKLOW et aL (1982) and are included here for comparison with the other data. Bacterial growth rates were calculated only from increases in cell numbers in incubated samples by linear regression of in N vs t, where N is the abundance of cells at time t (days). From these rate data, bacterial production was calculated as P = gNBc, (1) where P is the rate of production (Bg C 1-1 dq), B is the specific growth rate (day-1 ), Nis cell numbers (cells 1-1), and Bc is the mean mass of the bacteria (here, 1 × 10 -14 g C cell-l , or 10 fgC cell-l). Here we assume that the growth rate we observed in incubations following trap recovery is a good estimate of the mean rate in the trap during the deployment. This is likely to be a poor assumption in the ease of long deployments. Nonetheless the results should provide some indication at least of the scale of the processes revolved. If conversion efficiency of the bacteria (gC biomass produced) (gC substrate consumed) -1 is known, turnover times for the POC in the traps can be calculated from T- POC D (2) 449 Bacterial growth on POC in sediment traps where T is the decomposition turnover time (days), D is the decomposition rate (ttg C 1-j d-a) and POC is the concentration of POC in the sample (ttg C 1-]). D is calculated from D -- P(I ~ - e) (3) , where P is given in equation (1) above, and e is the conversion efficiency. For the calculations presented below we have made the conservative assumption that only the attached cells (defined above) decompose particles, so the rates P and D are scaled down by the proportion of attached cells given below. This and other assumptions are treated in more detail in the discussion section. RESULTS The sediment trap collections of particulate matter used in these studies were obtained from varying depths, temperature, and distance from shore during different seasons (Table 1, Fig. 1). Collections were made over time scales of hours (collections F 1 to F3) and months 77°39 ° 76"40: 75"41 • 74" 42 = .NEW. ..! • .. . . . III • ° ° . ; q ..• CAPE MAY S/ M I-3 25 0 !...... [ 25 t 50 , 75 ~ I00 i KILOMETERS Fig. 1. The continental shelf and slope of the Middle Atlantic Bight, showing areas where sediment traps were deployed. MI to M3 in the Baltimore Canyon area; F1 to F3 near the Hudson plume; M4 on the continental slope. See text and Table I for details of deployments. 450 H . W . DUCKLOWet al. Table 2. Sample Estimates o f total particulate flux and POC f l u x into sediment traps used in this study Total flux (rag m -2 d -I) Org. C. (%) Organic carbon flux (mgC m -2 d -1) FI* F2* F3* 12670 3580 2540 5.8 8.8 18.0 735 315 457 Mla Mlb Mlc 1239 266 105 7.0 7.4 86 8 M2a M2b M2c 768 107 111 6.0 6.1 - 46 7 - M3 1127 3.9 43 M4 1526 * Total fluxes as given in DUCKLOW el al. (1982). Organic carbon fluxes are based on %POC values from G. Rowe for these samples. (moored collections) (Table 1). The estimates of total and POC flux varied by factors of about 10 each. There was equal variability in the flux within and between deployments (Table 2). These estimates are similar to other fluxes sampled in the New York and MAB area (Rows and GARDNF.R, 1979; FAtXOWSKIet al., 1983). Flux estimates were greater in traps nearer to the bottom (Mla, M2a) and in shallower water (F 1 to F3), indicating the importance of resuapension of bottom materials in the lower water column. The sample from trap M3 may have had a low fraction of organic carbon (Table 2) because o f dilution from organic-poor sediments resusimaded from the sea floor and advected to the trap location. The variability in the POC data suggest a diversity of origins and fates of labile POC in sediment traps. Bacterial abundance and colonization of particulate matter in traps The density of bacteria in the trap samples (cells per mi of trap contents) was equal to or higher than bacterial densities in ambient waters (Table 3; DUCKLOWet aL, 1982). Absolute abundance levels in traps were high, ranging from 1.4 to 16.7 x 106 cells m1-1 in the bulk contents collected in traps. When these levels were normalized to themass of total particulate matter or to POC in the traps, values ranged from 0.8 to 331 x 10j° cells g-~, with highest values for total cells per gram POC, and lowest for attached cells per gram total sediment (Table 4). There were similar amounts of variability in all six parameters (total cells 1-1 or g-~ total particulates or POC; and attached cells 1-1 or g-I total particulates or POC) with coefficients of variation of 0.9 to 1.0 for the data in Table 4, and 1.6 to 1.9 for the data in Table 3. These bacterial densities are high (see Discussion and compare levels with values in Table 8) but bacterial colonization of particles in the traps was sparse. In the bulk contents of traps, particles were colonized by an average of 2.4 to 9:8 cells each (Table 3), similar to values for suspended particles in the water column (DUcKLOwand KIRCHMAN, 1983). Although a wide range for % attached cells was observed (15 to 88%, Table 3), 6 4 % of the particles in one set Bacterial growth on POC in sediment traps Table3. 451 Patterns of bacterial colonization of sedtment trap contents and surfaces Average number of Total cells % cellsattached attached ceilsper (10e cells ml-t) to particles particle FI F2 F3 2.9 1.5 1.4 66 24 15 3.0 2.4 3.6 Mla Mlb Mlc 8.0 5.5 4.6 59 54 15 4.5 9.8 1.5 M2a M2b M2c 7.2 5.3 6.2 43 42 48 4.1 3.6 4.2 16.7 92.8 46.2 88 64 20 4.8 21.8 13 0.3-1.5 <20 <4 Deployment M3 M3, wall washings M3, wall washings from inverted trap Mean ambient shelf water* * See, for example, I~CKLOWand KIRCHMAN(1983). Table 4. Bacterial densities in sediment trap contents expressed as cells per gram total particulate matter or per gram POC only Attached cells only Total cells (10' cells g-t) (109 cells gPOC -t) (10~ cells g-I) (109 cells gPOC -l) 33 45 55 570 510 310 22 11 8 380 130 40 Mla Mlc 25 180 360 2430 14 27 210 360 M2a M2b 40 200 670 3310 17 85 280 1390 M3 42 1070 37 950 Mean CV 78 0.9 1154 280 0.9 468 1.0 Deployment FI F2 F3 1.0 o f samples had 0 o r 1 cell per particle, and only 10 to 20% o f the particles h a d 10 o r m o r e cells (Fig. 2). These d a t a are based on a total o f 3530 particles in 36 samples from the M 2 time series (Figs 3 and 4). W a l l growth in traps One partial exception to these patterns concerns the slime scraped from the walls o f traps in mooring M3. T r a p walls were occasionally coated with layers o f slime o f varying thickness, which h a d high densities o f bacterial cells and high numbers o f cells per particle (Table 3). H.W. DUCKLOW et al. 452 W t.-- 40 r,tt/ 2 ! W ~ 2 _J n= 1071 M2c 40 Z I.t,I 20 ~J OI 0 v I 12-516.-10!11-49~'501 CELLS PER PARTICLE Fig, 2. Frequency of particle colonization by bacteriain incubated sedimenttrapcontents. ~ 6.6 0~ 6.4-] -- /s I t . _ _ _ e/ 6.2 6.0 ' 0 ¢:1 12 o b c 20 3o • • I i 2O f TOTAL C E L L S ~ ~"t~ ATTACHED C~LLS . . . . 20 ' ~o ' ~ ~ ~ ~ ~411 I' oo I ~o DAYS Fig. 3. Time courses of total bacterial and attached bacterial numbersin incubatedcontents from traps M2a,b,c. The data for attached cells refer only to particles (defmed in methods) held within the slime matrix, not to the total number of cells in the slime; however these m a y also be considered to be attached cells in the sense that they were not free cells suspended in water, like cells in interstitial spaces. It is possible the dense aggregations of bacteria in the slime attached to trap surfaces turn sediment traps into efficient reactors for P o e d e c o ~ . We have no data on the activity of these slimes. However slimes from seawater h ~ d s (m research vessels have extremdy high rates of [3H]thymidine incorlx)ration (H. DUCKLOWeI al., u n p ~ d Bacterialgrowthon POC in sedimenttraps 453 ,J 4 -~)~_~__e.---e-.. "'o .... ....... ==2---= -I O| "I I I I I I I I I I I 1 1o,, o 5.7 5.6 0 b 20 • C 30 1 2 I 0 portl¢los ml -I cellt partlc|e -I . . . . • , , 40 1 6 1 0 1 1 1 1 80 I00 DAYS Fig. 4. Time course of total particle concentrationand bacterial cells per particle for incubated contentsfromtraps M2a,b,c. Materialwas incubatedwithoutshakingat 5°C. data from R.V. Knorr, 1982). Comparison of the data from upright and inverted traps in M3 (Table 3) suggests that the slime can attract particles and bacteria from the bulk contents. The inverted trap had lower values for all parameters even though it had copious slime. Growth rates The growth of bacteria was monitored in different samples over different time intervals which were chosen to examine growth on the time scales over which sediment traps are often deployed. Growth rates were estimated over periods of hours (M4), days (F 1 to F3) and months (lVl1, M2). The time courses of bacterial numbers are shown in Figs 3 to 5 and 8, and discussed in turn below. Samples from moorings M 1 (data not shown) and M2 showed increasing numbers of total and attached cells (Fig. 3) over the first 24 days of the experiment, and also increasing number of cells per particle (Fig. 4). Most of the increase in total cells could be accounted for by increases in attached cells. For example, the total and attached cells in sample M2a increased by 2.9 and 2.6 x 106 cells ml-~ , respectively. This suggests that bacterial growth over this time scale was sustained by the decomposition of particulate organic matter, rather than at the expense of dissolved material in the water in the samples. Over the same 24-day period, the total number of particles increased (Fig. 4), possibly because of fragmentation due to biological activity. Between days 24 and 99, values of these properties stabilized, declined, or varied irregularly. Note especially the precipitous decline in attached cells in M2a and M2b between days 24 and 73 (Fig. 3). This may have been due to predation or autolysis but not simply detachment, because free cells also declined. Growth was measured over periods of 3 to 5 days in samples F 1 to F3 aboard ship immediately following recovery. In sample F2 (Fig. 5B), growth ceased after 1.2 days. In sample M4, we observed slow growth between 2 and 10 h of incubation (Fig. 8B). Note that cell numbers increased and then levelled off on all 3 time scales of observation. This indicates the importance of investigating growth processes over a variety of time scales relevant to sediment trap deployments. 454 H.W. DUCKLOW et al. ~o~o A 9oo 750 ~,' soo- ~1500 I I i I 40 50 I 60 I I I I I I0 20 i 30 __. 9.zo" o g -- 9. l - 9.0-~ 8.9 [ 0 70 hours Fig. 5. Time course of: (A) incorporation rate (not incorporation) of [3HJthymidine; and (B) bacterial numbers in incubated contents of trap F2. The mUnlde was i n c u ~ for 48 h on ship irn~y following recovery, after a 24-h deployment. In this experintent, alaRge (ep. 1 !) ,ample was incubated, and 10nd aliquots were withdrawn periodically for AODC or [*H]thymidine incorporation assays. Growth rates and generation (doubtinlOtimes calculated from chattBm in cell nmnbers were generally slow, ranging from 0 to 0.33 d -1 and 2.1 to >77 days, respectively (Table 5). In general the highest rates were found when growth was monitored over shorter time scales, and also in the freshly captured materials from the floating traps. These samples had the highest organic matter contents (Table 2), and were derived from a sedimenting Skeletonema eostatum bloom following a storm (MALONEet al., I983). Growth was very slow when Table 5. Sample SpeeO~c growth rates of bacteria in sediment trap contents, estimated from dmnges in cell number Incubation temperature (*C) Rate (d-O Generation time (d) F1 F2 F3 3 3 3 0.33 0.09 0.34 M la Mlb Mlc 5 5 5 0.009* 0* 0.015" 77 46 M2,a M2b M2c 5 5 5 0.012" 0.009* 0.013" 58 77 53 M4 15 * During the first 24 days (Fig. 3). 0.21 2.1 7.7 2.0 3.3 Bacterialgrowthon POC in sedimenttraps 455 integrated over longer periods (the M 1, M2 samples). These rates could be more representative of bacterial activity in traps deployed for longer periods. However the contmts of trap M4 retained the capacity to support measurable growth over hourly time scales ¢ven after a 99-day deployment and 6 months of storage (Fig. 8B, M4). The prolonged growtbsustaining quality of sediment trap contents is discussed with respect to macromolecular synthesis in the next section. Incorporation o f nucleic acid precursors and macromolecular synthesis Bacterial activity related to cell division was also studied by monitoring the inc3rporation of the nucleic acid precursors [3H]thymidine (3H-TdR) and [3H]adenine into macrcmolecular fractions of sediment trap contents. In the incubation experiment with samples fror~ the floating traps, the rate of total [3H]TdR incorporation into cold, 5% trichloroacetic add (TCA) extracts increased by a factor of 10 in 2.5 days (Fig. 5A), at a rate of 0.90 d "~. Thi2 was well in excess of the growth rate computed from observing changes in cell numbers (0.0~ d -t , Fig. 5B and Table 5). These rates should be equal if [3H]TdR incorporation into DN,A and cell division are closely coupled (KmcHMAN et al., 1982). There are several differexR re.sons for this discrepancy. It is possible that in these samples, as in most seawater samples (IUHRMAN and AZAM, 1982), [3H]TdR was incorporated mostly into DNA, but that this prccess was not coupled to cell division over the time scale of this experiment (KIRCHMAN et ~L, 1982; DUCgLOW and HILL, 1984). Alternatively the [3H]TdR may have been incc'porated primarily into RNA or protein, with only small amounts going into DNA. If this ~,ere the case, the increase in the rate of [3H]TdR incorporation into DNA alone may h~ve been closer to the growth rate. We studied these processes in greater detail by determaing the rates of specific incorporation of [3H]TdR and [3H]adenine into DNA, RNA, am protein separately in the contents of trap M4. Although the sample was more than six months old at the time of the experimnt, the incorporation of both [3H]TdR and [3H]adenine began immediately, and boh were approximately linear over a 60 to 90-rain incubation period (Figs 6A and 7A. Total incorporation rates of both compounds were high (Table 6). None of the incorporatim rates showed a tendency to saturate, even at 50 nM levels of added labelled substrate (Figs6B and 7B). This behavior suggests that even after six months of storage at low temperature, ~actetia were not dormant, and that the sediment trap material retained the capacity to suppot acti~ Table 6. Average specbqcity of labelling by exogeneous [3H]thymidine and PH]adenine of macromoleadarfractions of sediment trap material Incubation times were 60 mtn Rate of labeUingof fraction _+s.d. pmol 1-~ h-t (% of total) [~HI-Labelledsubstrate Thymidine* Adenine* RNA DNA Protein Total 13.1 + 1.2 (63) 3.3 + 0.7 (16) 4.3 + 1.3 (21) 20.7 (IOO) n=6 n=7 n=8 561 + 133 (83) n=6 82 _+20 (12) n=9 34 + 9 (5) n=10 * Both substrates addedto 5 nM finalconcentration. 677 (lOO) 456 H . W . DUCKLOWe t aL 25- 20- RNA T / y :O-- ~ IO-q I o. o. I0- 5- 5- ! DNA 0- I 0 30 60 ,o 90 minutes Fig.6. ~o I ~o ~o ~o subtrote conc., InM): (A) Time course Of specific incorporation of [3Hlthymidine into ~ N A and RNA, for con- tent of trap M4 followin86 monthsof storage at 5"C. (B) Effect of initial [ H~yn',idine ~centrationon incorporation into DNA and RNA for contents of M4. In th--,~,ee ~ m e n t s , added to each sample at zero time, but after 6 months of storage. A isotope was B RNA P 900- // // 600- ~C0- // T. //// i ).500!000- / II °E o.. / 300- RNA 5500- // 750- 500I000- DNA DNA 500- x~ I I [ 30 I I 60 90 minutes FiB 7. 0 I I I 0 substrate conc., (nMI (A and B) as in Fig. 5 for trap sample M4, but employing [3H]adenine as substrate. In Fig. 6A, the 60-rain sample was lost. bacterial synthesis of cellular constitutents. However these results also suggest that growth, estimated from D N A synthesis, was slow compared to R N A or protein synthesis. The ncorporation rate of each precursor into D N A was only 12 to 16% of its total incorporation into cold 5% TCA insoluble cell fractions (Table 6). In the upper water column usually 60 to 8096 of [3H]TdR in such extracts is in D N A , with only 20 to 40% in R N A and protein (FUHRMANand AZAM, 1982). In this experiment R N A synthesis accounted for 63 to 83% of tae total incorporation, and more [3 H] TdR was incorporated into protein than D N A in a 60-nin period (Table 6). These patterns of [3H]TdR incorporation are not consistent with our current understanding of pathways for the incorporation of exogenous TdR (KoRNSE~, 1980). HANSON and LOWERY(1983) also observed that in bacteria from below 1000 m u! to 8096 of [s H]TdR incorporated was in protein. We do not know the reason for these cont-asting incorporation patterns. In another incubation experiment (trap M4) similar 457 Bacterial growth on POC in sediment traps to those shown in Fig. 5, the incorporation rate of [3H]thymidine into DNA was essentially constant over a 10-h incubation period (Fig. 8A), while the incorporation rate of [ 3H]thymidine into RNA rose over the fu'st 4 h, then levelled off. These results indicate that in the samples we have studied, the total incorporation of nucleic acid precursors into DNA, RNA and protein are not closely coupled to cell growth (as evidenced by different slopes for the time course of radioactivity and cell numbers) because most of the label is incorporated into RNA and protein and not into DNA. The main significance of this observation for the present study is that even though bacterial growth per se may be low, bacterial metabolism as a whole may be significant. With this point in mind, we proceed to estimate the decomposition of POC in our samples from bacterial growth rates. Bacterial production and POC decomposition We have calculated attached bacterial production and decomposition rates from the growth rates listed in Table 4, the abundances of attached cells (Table 2), and mean cell masses of 10 fgC cell-~ (equations 1 to 3). The resulting values (Table 7) are conservative for several reasons, which are discussed below. Attached bacterial production ranged from 0.1 to 6.3 ttgC 1-1 d-~ (mean 1.2 + 0.9, n = 7), which is similar to values for attached bacteria in the Hudson River plume at 3°C (DucKLOWand KIRCHMAN,1983). The turnover times of POC in the traps due to in situ bacterial decomposition alone were long, averaging 1800 + 600 days (Table 7). The significance of this finding is discussed below. DISCUSSION Sediment traps capture a population of particles which are presumed to be relatively rare in the water column and may not be efficiently sampled by water bottles (SHELDONet al., 1972; SASAKIand NISHIZAWA,1981). Therefore, we do not have direct methods for sampling these large, rapidly settling particles and measuring their decomposition rates. At present we need to extrapolate such estimates from measurements made on collections of particles collected in and recovered from sediment traps (DUCKLOWet al., 1982; KARLand K~AUER, 1984). The accuracy of these extrapolated estimates will be greatest when derived from traps Table 7. Bacterial production estimates and decomposition turnover times for sediment trap POC Attached bacterial prod. (p.g C i-~ d -~) Decomposition rate (IJ,g C 1-l d -~) POC concentration ~ g C !-l) POC turnover time (days)~ FI F2 F3 6,3 0.3 0.7 57 2.7 6.3 5106 2923 4571 90 1082 726 Mla Mlc 0.4 ~1 3.8 0.9 22284 1889 5829 2100 M2a M2b 0,4 0,2 3.6 1.8 10708 1598 2974 888 M3 1.5" 15908 1223 Sample 13 * Assumed growth rate of 0.01 d -~. ? Calculated from equation (2) assuming bacterial conversion efficiency of 1096. 458 H.W. DUCKLOWet al. deployed for short periods. Since the rates of decay of particulate organic matter decline as particles age (FENCHI~Land JORGENSEN, 1977) estimates from long deployments are likely to be underestimates of the 'true' decomposition rates. Measurements made on sediment trap collections also yield data on the related problem of particle decomposition rates inside the traps following capture. The length of the trap deployment also is a factor influencing these estimates. As particles age and decay, an increasingly refractory suite of organic compounds remains in the traps, and decay rates decline (GARDNERet al., 1983). The effect of deployment time, and the interaction between vertical flux, or collection rates and varying decay rates has been modelled previously (GARDNERel al., 1983) and will not be treated in detail here. However, the relationship between the decay of particles in the water column, and their decay inside traps will be discussed below. In our study, traps were deployed for either 1 or ca. 100 days, and trap depths ranged from 10 to 1000 m in water columns 20 to 1200 m deep. Later, we will assess decomposition of sediment trap particle collections in two contrasting regimes: short deployments in shallow water, and long deployments below the euphoric mixed layer. Our studies concern particles originating and collected over the continental shelf and upper to mid-slope region, not from the deep ocean. Particles, verticalfluxes, and associated biological activities in open ocean areas differ qualitatively and quantitatively from shelfslope regions (EPPLEY, 1980; WALSa, 1983). For instance, in midwater depths in the open ocean, chemoautotrophic production associated with particles near the oxygen minimum may elevate microbial activity in traps without directly affecting decomposition of particles (KARL and KNAUER, 1984). Our conclusions will not necessarily apply to similar oceanic regimes. To our knowledge, there has only been one other study in which bacterial colonization of sediment trap particles was measured. ITURRIAGA(1979) found that at 10 to 18-m depths in the Baltic Sea, there were 0.2 to 0.6 x 109 cells per gram of POM in traps deployed monthly over the course o f a year. Our values are much higher, ranging from 300 to 3000 x 109 cells g-i POC (Table 4). We can further assess bacterial colonization of large particles by comparing our data to bacterial abundances in surface sediments, which are likely to be derived in part from deposition of the vertical flux. Some values for total bacterial abundances per gram of sediment are presented in Table 8, for comparison with the frst column of data in Table 4. Even the highest values in rich salt marsh sediments (50 to 70 x 109 cells g-') are only equivalent to the lower values we measured (20 to 200 x 109 cells g-'). Since the organic content of such sediment is likely to be around 1 to 10% (RusLEE and DORNSEIF, 1978), these levels translate to 500 to 7000 × 109 cells g-' POC, which is similar to our corresponding values. NowrsKV (1983) carefully sampled the interfacial sediments most likely to be similar to falling particles, in an environment similar to some of our study areas. He found only 1 to 10 × 109 cells g-t, about an order of magnitude lower than our estimates. While such measurements are not directly comparable, they indicate that very high numbers of bacteria are associated with the contents o f sediment traps. High numbers of bacteria associated with material in sediment traps suggest a great potential for rapid decay and a potentially major role for bacteria in particle decay. But our findings also suggest that this potential may not be realized, or perhaps that in most cases, our observations missed the period of rapid bacterial decomposition (Table 7). On average, only about 50% of the particles were colonized, by a mean of ca. 1 to 10 cells per particle (Table 3, Fig. 2). The bacterial abundance data in Tables 3 to 5 suggest that bacterial carbon is probably a small fraction of the total POC in the traps. Using a mean value of 10-'4 gC Bacterial growth on POC in sediment traps Table 8. Bacterial content of marine sediments. Unless otherwise noted, values are for surface (0 to 1 cm) of sediments, and per gram total sediment 10~ cells g-i 0.8-22 69.7 60.1--66.3 38-54 3.3-8.8 4.2-6.0 2-100 0.3--10 0.4-0.5 1.2-2.3 0.4--2.7 1.2-1.7 1.1--9.7 0.2-0.6 0.16---0.66 16-310 459 Sediment type Spartina marsh, North Carolina Spartina marsh, North Carolina Low and high marshes, Rhode River, MD Sippewissett marsh, MA Subtidal mud, Newport River estuary, NC Intertidal mud flats, Nova Scotia Subtidal sediments, Denmark Intertidal sand flat, Nova Scotia Shallow subtidal sediments, Shark Bay, Australia Stromatolite mats, Shark Bay, Australia Sandy beaches, Baltic Sea Seagrass sediments, Queensland, Australia Sediment-water interface, Halifax Harbor. Nova Scotia, 22 m deep Sediment trap contents in Baltic Sea at 10and 18m, per g POM Suspended POC, Hudson River plume of New York Bight Reference RUBLEE(1982a) RUBLEEand DORNSEIF (1978) RUBLEE(1982b) RUSLEE (1982b) RUSLEE (1982b) RUSLEE (1982b) C AMMEN(1982) DALE (1974) MORIARTY (1983) MORIARTY (1983) MEYER-REIL et al. (1978) MORIARTY and POLLARD (1981 ) NOVlTSKY (1983) ITURRIAGA(1979) DUCKLOW and KIRCHMAN (1983) cell-m, data in Table 4 indicate that attached bacterial carbon is a maximum of only 3.3% of the POC (trap M2b, Table 4). Therefore, unless the bacteria are growing very rapidly, their impact on the POC in the traps could only be minor. The growth rates we observed were relatively low (Table 5), though we emphasize that when rates are measured over longer periods (e.g., traps M 1, M2) they are lower than shorter term estimates. Nonetheless, even the faster estimates are low compared to growth rates measured for planktonic bacteria in the water column. This result may be because bacteria in the water column are growing on fresher, more labile material than bacteria in traps. These rates usually average 0.5 to 2 d-~ (DucKLOW, 1983). Since the bacteria were a small fraction of the total POC in the traps, and grew slowly, the decomposition due to bacteria was also slow (Table 7). Our decomposition rate estimates are subject to several uncertainties. Chief among these are the accuracy of the growth rates, and the conversion efficiency of the bacteria. For the estimates in Table 7, we took the rate data at face value, and assumed conversion efficiencies of 10% (i.e., 0.1 g of biomass carbon produced per gram POC consumed). Although planktonic bacteria utilizing dissolved compounds like amino acids and mono- or oligosaccharides convert DOC into biomass at efficiencies of 50 to 90% (WmUAMS, 1981), attached bacteria converting POC into biomass are probably less efficient. This is because they must synthesize and release extracellular enzymes to degrade their substrates which are more refractory than low molecular weight DOC. NEWELLet al. (1981) found that bacteria growing on phytoplankton debris had conversion efficiencies averaging 10%. POM~gOY et al. 0 9 8 4 ) found that bacteria converted pelagic fecal material at 10 to 20% efficiency. It is likely that phytoplankton debris and fecal materials make up an important fraction of the materials in our samples. There is not a convenient direct method for estimating this parameter in our samples. However, we observed that bacterial populations in samples M4 were synthesizing RNA and protein, but not D N A rapidly (Table 6 and Figs 6 to 8). Cells 460 H.W. DUCKLOWet al. R~A 1214 t _-/ /x~------°'--'-~"o RNA IO-c,--jr -6 i ~~~=~_~-.-~ o i i 1 DNA I IO.4- B T o_ =_o T 10.5- 1 i hours Fig. 8. Time courses of: (A) specific incorporation rates of [3H]thymidine into D N A and RNA; and (B) bacterial numbers, for incubated contents o f trap M4. This sample was incubated for 10 h following storage at 5°C for ca. 6 months. Each rate measurement was derived from a 30-ram assay. This experimentis similar to that described for Fig. 5. were active but not growing very fast. It is unlikely that bacteria in our samples were more efficient than 1096, though they could have been less efficient. As we suggested above, some of the growth rates we measured are likely to be underestimates, since predation in the samples, low temperatures, and the time scales of the trap deployments and measurements would all interact to lower the observed rates of increase in cell numbers. Finally, even free bacteria probably contribute to particle decay in traps. For these reasons, the decomposition turnover times presented in Table 7 are likely to be conservative. The estimates of turnover times can be scaled down by assuming that growth rates are higher, conversion efficiencies are lower, and that free bacteria also contribute to particle decay. For exam#c, by assuming that in M l a , the free and attached cells all degraded POC and grew at 0.05d -t , the decomposition turnover time is reduced to 620 days. This is still a tong time, and helps to explain why slope sediments off the MAB have high organic carbon content (WALSH, 1983). Other reports suggest that bacteria do grow in sediment traps and show that preservatives inhibit bacterial growth. LEE et al. (1983) analyzed samples from a time-series sediment trap deployed for 3 months for muramic acid, a compound unique to the cell walls of gramnegative bacteria. Muramic acid was detected in the early samples which were closed and incubated in the trap the longest, but not in later samples which were in thetrap for a shorter time. Because of the presence of bacteria in the older samples they assumed that sodium azide added to the trap at the time of deployment had diffused away. They did not detect muramic acid in other experiments where deployment time was short and no preservatives were used (LEE and CRONIN, 1982), in a 2-week deployment with chloroform used as a preservative (LEE etaL, 1983), nor in a 61-day deployment in which sodium azide was continuously leached into the sample (LEE etal., 1983). In all these experiments, bacteria undoubtedly entered the traps in the vertical flux, but their biomass was probably too low for detection by the chemical method used. Bacterial growthon POC in sedimenttraps 461 There exist several data sets for comparison with our estimates of bacterial POC decomposition. ITUR~AGA (1979) and LORENZENet al. (1983) noted decay rates of 0.03 to 0.08 d-I for materials in traps deployed at shallow depths for short periods (i.e. turnover times of 12 to 30 days). GARDNERet al. (1983) found that mixed zooplankton held in deep traps for 125 days had a turnover time of 235 days (0.004 d-m). They also formulated a model of trap collection and decay rates, and using data from LEEet al. (1983) suggested that POC turnover times in deep traps deployed for longer periods should be on the order of 100 to 1000 days, within the range shown in Table 8. Several lines of evidence indicate that particles in the surface layer of the ocean are more rapidly decomposed. From observed changes in the composition of particulate amino acids in a series of traps, LEE and CRONIN(1982) estimated that 80% of the primary production was decomposed above 14 m in coastal waters off Peru. EEPPLEYand PETERSON(1979) suggest that in most of the ocean 80% of production is recycled in the surface layer. If one assumes that most primary production results in large particles (Diatoms, fecal pellets, or scavenged aggregates) on time scales of a few days, then these findings demand that decomposition rates must be close to 0.5 to 1.0 d-~ in the surface layer. This is because large particles sink at rates of at least 10 m d -~, and probably as fast as 50 to 100 m d-~ (DEUSER etal., 1981; LORENZENetal., 1983). Therefore, 80% of the large particle flux must be recycled in the few days it takes to leave the 20 to 100 m deep surface layer. EPPLEYet al. (1983) estimated from POC stocks and regenerated production rates (NH~-supported primary production sustained from recycled PON) that recycling times in the Southern California Bight were 1 0 _ 5 days. Comparison of these two sets of estimates suggest two time scales for POC decomposition: 1 to 10 days for POC in the surface layer, and 100 to 1000 days for the remaining POC which sinks out of the surface layer. We expect that the fraction of POC not recycled in the first few days is less labile than the 80% which is recycled, so a reduction in decay rates as particles fall into deeper, colder water is not surprising. LORENZENet al. (1983) have observed lower carbon loss rates in deeper sediment traps. However, FUHRMANand AZAM(1983) have observed that attached bacteria collected below the mixed layer have temperature optima characteristic of surface layer planktonic (free) bacteria. They assumed that these attached cells were derived from the surface layer on falling particles. Thus we are not yet certain of the time scales for the transition from high to low growth and decomposition rates. Our observations on decomposition rates are more characteristic of more slowly decomposing POC from deep or cold water. Our estimates for decomposition turnover times average 1500 + 350 days, with the two extreme values removed (Table 7). We believe these may be overestimates of the true turnover times, but it is not possible to judge how conservative our values are. If we assume that these values are realistic, we must conclude that bacteria acting alone are not the principal agents of particle decomposition within sediment traps. The logarithmic mean of the GARDNER et al. 0983) range is 300 days, so we suggest that bacteria alone contribute a minimum of about 20% of the total decomposition of POC in sediment traps. It seems likely that the main fate of POC is to be ingested rather than decomposed. This is not an unreasonable conclusion. Since only about l0 bacterial cells colonized the average particle (Table 3), and bacteria were only about 1% of the POC in the traps, it is unrealistic to imagine that rather small bacteria (ca. 10fgC cell-~ ) which only sparsely colonize large particles, are major agents of particle decomposition. In comparison, bacteria account for about 2 to 4% of the POC in marine sediments (CAMMEtq, 1982; Rt:BI~EE, 1982b). 462 H.W. DUCKLOWet al. Our measurements did not take into account the decomposing activities of fungi, protozoans, and micrometazoans. Larger suspended particles like organic aggregates (CARON et al., 1982) and marine snow from sediment traps (SILVERand ALLDREDGE, 1981) have dense and diverse collections of these organisms. The respiratory activity of phagotrophic proto- and microzooplankton can directly mineralize ingested particles, and grazing reduces particle sizes, thereby increasing surface areas for bacterial attachment (JOHANNES, 1965). Finally, we note that sediment traps sometimes have accumulations of gel-like material coating trap walls, which contains high densities of bacteria and densely colonized particles (Table 3). Our mooring experiences indicate that the abundance of slime decreases rapidly with depth on PVC, metal and glass surfaces, and seldom occurs below ca. lO00 m. Build-up of slime was greatly reduced in sediment traps by the use of antibiotics imbedded in a silica gel attached inside of traps (DYMONV et al., 1981). Based on uptake rates of 14C-labelled glutamic acid, however, the biological activity in sediment in poisoned and unpoisoned traps was similar. This slime may render sediment traps analogous to biofdmcoated stones in the trickling filters used in sewage treatment (LARIVX~RE, 1972). If such a function is common in traps, the role of bacteria may be greater than we have suggested here. It is clear that few unequivocal generalizations concerning POC decomposition are warranted at present. But most of the data suggest that bacterial decomposition of large particles and suspended POC in the upper and lower water column is stow, while total decomposition rates may be more rapid. To resolve the processes of POC decomposition in the water column, bacteriologists, protozoologists, and geochemists need to conduct sampling and experimental techniques with close attention to the diversity of particle types and time and space scales on which the processes proceed. In this study we have extended several earlier observational approaches to sediment trap collections from several locations, and have confLrmed earlier bacteriological observations that bacterial involvement in POC transformation appears to be minor on long time scales. This conclusion should be examined with caution until further data become available. Acknowledgements--Sediment trap deploymentswere supported by DOE Contracts DE-AC02,76-EV02185Eto LDGO ~ DE-AC02-76-CH00016 to BrooldmvenNational Laboratory and by BLM Contract AA851-CTO,59 to LDGO. We are grateful to P. Biscaye, T. Malone, G. Rowe; J. Walsh and C. McCulloch for scientific assistance, and to the officers and crews of R.V. Knorr and=R.V. 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