4.1Shelf sand – a physically dynamic habitat
The data we collected indicate possible adaptations of a benthic bacterial population to the hydrodynamic forces acting on a sandy shelf sediment in the Middle Atlantic Bight. Unlike most depth distributions of bacterial cells in sediments less affected by waves and bottom currents, two of our four profiles showed maximum abundances below the sediment surface (Fig. 2). We hypothesize a loss of cells from the most exposed upper layer due to hydrodynamical stress. This view, though corroborated by observations in an intertidal sandflat , lacks direct support by our restricted set of hydrodynamic data (Table 2) and the snapshot nature of our sampling.
Interstitial and interfacial water flows are common in permeable shallow water sediments [12–18]. Bacteria attached to sand grains should be less susceptible to removal and are therefore expected to dominate such sediments. Our finding that free-living cells comprised less than 0.2% of total bacteria in our shelf sand supports this supposition. Likewise, bacteria attached to particles of the fine fraction were of minor abundance, as the ‘coarse fraction’ was mass dominant, held the major share of the sedimentary bacteria (Fig. 7), and was responsible for the spatiotemporal variability of cell numbers. In spite of possible nutritional disadvantages, the less mobile microhabitat of coarser grains apparently can sustain a population of sand bacteria.
4.2Sand bacteria and their biogeochemically dynamic habitat
In permeable sediments, physical and chemical interfaces move frequently, and benthic microbes experience highly variable biogeochemical conditions. The activity of extracellular hydrolases, considered rate-limiting in the microbial degradation of organic matter in waters and sediments [57–59], may be crucial in making optimal use of the intermittently supplied POM. The rates of biopolymer hydrolysis measured in LEO-15 sand were in the same range as those reported from sediments with relatively low organic content or comparable hydrodynamic regime [59–61]. In a coarse-grained Antarctic sediment exposed to strong bottom currents, the activity of aminopeptidase exceeded that of β-glucosidase only by a factor of about 10 , similar to measurements in fresh diatom aggregates  as well as in our shelf sand. Benthic microbes in nutrient-poor habitats may rely on high peptidase activity to obtain sufficient amounts of organic nitrogen, whereas ample supply of fresh POM implies less of a requirement for protein hydrolysis. The relatively weak preponderance of peptidase compared to β-glucosidase activity in our study suggests that the benthic community at LEO-15 is not severely limited by nitrogen. The seasonality of exoenzyme activity (Fig. 5) and correlations with algal material, POC and DOC (Fig. 4) favor the scenario of limitation by organic matter quantity rather than its nutritional quality. The overall low activity in August 00 may indicate that episodes of very strong bottom currents and wave action (Table 2) periodically remove extracellular enzymes and/or compounds that are involved in regulating their activity.
Evaluating the metabolic status of bacterial cells or populations is largely based on criteria like membrane integrity, cellular reducing potential, specific enzyme activity, or rRNA content . Methodical biases include differences and interactions between natural and tracer substrates with respect to concentrations, uptake and transport rates, or substrate specificity. In LEO-15 sand, the relative size of the metabolically active subpopulation of benthic microbes, as inferred from INT reduction, was closely related to organic matter (and we suspect oxygen) supply to the sediment. Both the total abundance of bacterial cells and the proportion of INT reducing cells changed with time and sediment depth (Fig. 2), mainly in the coarse fraction. With the fine fraction holding relatively large shares of algal cells (Fig. 7), it can be assumed to represent the episodic input of suspended POM to the sediment community throughout the year. By contrast, the coarse fraction and associated microbes represent a matrix that receives these POM inputs and responds to intermittent supply. This view is supported by depth profiles of INT reduction (Fig. 2) that often paralleled those of algal cells, [DOC] or exoenzymatic activity. Reversible adsorption of organic material to mineral surfaces [64,65] may buffer its episodic influx towards microbial utilization. Additionally, oxygen supply plays a key role in sediments like the LEO-15 sand, because the stimulation of benthic mineralization by oxygen has been found strongest in coarse-grained, organic-poor sediments . At all times sampled, current and wave impact (Table 2) on the permeable bottom facilitated advective flows that could enhance the influx of oxygen and organic compounds, and thus, intensify POM degradation (Reimers et al., in preparation).
Thus, pool sizes in the course of POM degradation, exoenzymatic turnover rates and the physiological state of cells paint a coherent picture of the microbial population of a permeable shelf sediment as being versatile with respect to the mechanical and biogeochemical dynamics of their habitat.
4.3Bacterial community composition of a permeable subtidal sediment
The molecular analyses of cells from LEO-15 sand indicate a microbial community almost exclusively composed of planctomycetes and members of the Cytophaga/Flavobacterium cluster (Fig. 6).
Many cultured species of the Cytophaga/Flavobacterium group produce exoenzymes and aerobically degrade a variety of high molecular mass organic compounds [67–70]. FISH studies proved them a dominant group in marine bacterioplankton [69,71,72] and highly abundant also in marine sediments [27,45]. Throughout our study at LEO-15, the Cytophaga/Flavobacterium cluster was dominating the benthic bacterial community, but occurred in much lower concentrations in bacterioplankton samples from the overlying water (Fig. 6). With the water column differing drastically from the sediment physically and chemically in many respects, differences in microbial community structure are not surprising. However, it is also reasonable to propose that the pore space of permeable sediments may hold a microbial subpopulation similar to that of the water column, since it is tightly coupled to the bottom water by interfacial and interstitial water flows. Investigations to test this hypothesis were beyond the scope of our present study.
Planctomycetes are a very widespread group of chemoheterotrophic bacteria typically degrading polymeric substances aerobically, but they have also been detected in anoxic zones of marine sediments . Planctomycetes were also a major group in the benthic microbial community of the permeable sand we studied (Fig. 6). Together with the Cytophaga/Flavobacterium cluster, they formed a benthic community of bacteria likely to degrade biopolymers. Indeed, we measured considerable potential rates of carbohydrate and peptide hydrolysis in the LEO-15 sediment (Fig. 5). Although both POM supply (Fig. 3) and exoenzymatic activities (Fig. 5) varied markedly with depth and seasons, our set of FISH probes failed to detect corresponding changes in microbial community composition (Fig. 6). Adaptation to spatiotemporal dynamics of our sandy sediment could have been achieved by changes in cell number or activity that affected all bacterial groups equally. An alternate explanation is that the limited number of samples, relatively wide statistical variation, and the broad specificity of the probes we used were not sensitive to possible differences.
Phytoplankton blooms have been shown to be accompanied by pronounced changes in bacterioplankton abundance, productivity, exoenzymes, and community composition [32,73,74]. The sediment surface sample taken during the July 01 bloom situation was unique with respect to detectable numbers of γ-Proteobacteria (Fig. 6), a group with many copiotrophic members . Moreover, the ratio between CF 319a and PLA 886 positive cells was 2.4 in July 01 compared to 1.7–2.1 in the other months (Fig. 6). According to ongoing studies by Llobet-Brossa et al. (personal communication), high CF:PLA ratios are frequently found in sediments rich in labile organic carbon. In our case, they might reflect a community response to the ample supply of fresh algal material.
The general dominance of planctomycetes and Cytophaga/Flavobacterium relatives, both preferably attaching to particles [68,70,71,76,77], agrees well with our finding that less than 0.2% of the sand bacteria were discovered free-living in the pore water. Proteobacteria, that are typically free-living , occurred in low abundances. Some isolated strains of planctomycetes are capable of oxidizing organic substrates via nitrate reduction , and members of the Cytophaga/Flavobacterium cluster have been shown to degrade monomeric organic compounds in coastal waters near our study site . Hence, these groups could comprise both the hydrolyzing and the mineralizing subpopulation of microbes inhabiting LEO-15 sand, and some species might even be able to run both kinds of heterotrophic metabolism. Species that both ferment and mineralize, however, may fill the niche less successfully than a consortium of mineralization specialists, e.g. Proteobacteria, and fermentation specialists.
Considering the general oxic appearance of our cores and the penetration of oxygen deep into the permeable sand (Reimers et al., in preparation), sulfate reducers are not necessarily expected to be active, but have repeatedly been observed in high abundances, mostly Desulfovibrio sp., in oxic marine sediments . We failed to detect them at LEO-15; due to the narrow specificity of the probe, however, we cannot exclude the presence of other sulfate reducing bacteria in this sediment. With the molecular methods applied to our sand still requiring thorough revision and improvement, we wish to have our FISH data viewed conservatively and interpreted with caution.
In conclusion, we have identified and quantified the major phylogenetic groups represented in the active microbial community of a permeable shelf sediment. This ecological study also gives evidence for the bacterial population of marine sands responding to the biogeochemical variability of their habitat. Hydrodynamical impact on the benthic community appeared probable, but remains to be proven by further investigations.