Environ. Sci. Technol. 2001, 35, 2275-2281
Incipient Erosion of Biostabilized Sediments Examined Using Particle-Field Optical Holography K E V I N S . B L A C K , * ,† H O N G Y U E S U N , ‡ GARY CRAIG,‡ DAVID M. PATERSON,† JOHN WATSON,‡ AND TREVOR TOLHURST† Gatty Marine Laboratory, University of St Andrews, East Sands, St Andrews, Fife, KY16 8LB Scotland, and Department of Engineering, King’s College, Aberdeen University, Aberdeen, AB24 3UE Scotland
Laser holography allows images of three-dimensional space at ultra-high resolution to be recorded onto photographic plates. Recorded scenes can be “replayed” with a second laser beam into free space and optically “interrogated” using either a microscope or a camera by sequentially focusing on increasing distances from the hologram in the field of view (optical sectioning). From these sections, information on the relative locations and orientation in space of suspended particles as well as the morphology of particles can be obtained. This paper examines the utility of “in-line” laser holography to discriminate the size and the morphology of sand particles eroded under turbulent shear flow during benthic sediment transport. The influence of a commercially available adhesive polymer (xanthan gum, derived from the bacterium Xanthomonas campestris) on sediment stability and resuspended particle morphology is described. The major implications for carbon and sediment cycling within estuaries are highlighted.
Introduction Since the early pioneering work of Carder et al. (1), laser technology has been used to provide accurate, high-resolution images of underwater objects (e.g., refs 2 and 3). This paper describes the application of laser holography to the study of incipient sediment erosion. Laser holography allows images of three-dimensional space with retention of all paraxial and perspective information to be recorded permanently onto photographic plates. This presents considerable advantages over other imaging technologies occasionally used in sediment transport contexts (e.g., refs 4-6) or other visual inspection techniques, such as stereophotography. In addition, it offers an extremely high resolution of individual grains (∼10 µm) over a depth of field that may be several tens of centimeters (7). The recorded scene can be “replayed” with another (continuous) laser beam into free space and optically “interrogated” using either a microscope or a camera by sequentially focusing on increasing distances from the hologram in the field of view (optical sectioning). From these sections, a model of the subject volume, and hence the relative locations and orientation in space of suspended particles, * Corresponding author e-mail:
[email protected]; telephone: 011 44 1334 463442; fax: 011 44 1334 463443. † University of St Andrews. ‡ King’s College. 10.1021/es0014739 CCC: $20.00 Published on Web 05/04/2001
2001 American Chemical Society
can be generated. This permits detailed investigation of the processes of erosion and deposition of both sand and mud in ultra-high spatial detail. The nature and properties of particles in suspension in aquatic environments has been extensively studied (e.g., ref 8) but less is known regarding the nature of deposited particles as they are entrained through fluid drag, especially where those sediments are cohesive (muddy) in nature. To an extent, the nature of resuspended sediments will be a function of the grain size, density, and mineralogy of the constituent grains (9). However, research in the last two decades has shown significant post-depositional mediation of sediment properties by benthic flora and fauna (10, 11), particularly in the intertidal zones of estuaries. Surface-dwelling photosynthetic microalgae, in particular, are known to secrete adhesive polymers (extracellular polymeric substance; EPS) that bind the mineral grains together in a gelatinous matrix (12). In certain cases, this has been observed to have substantial, ecosystem-wide consequences (e.g., ref 13). A major focus of this work is to examine the potential role of benthic microorganisms in moderating the types of particles/ aggregates that are eroded by moving seawater flows and to investigate the relationship between near-bed shear forces at the point of erosion and the distribution of eroded particle size and morphology. At this stage, we have used a simplified approach and used isolated microbial polymer exudates only rather than microbial assemblages to modify particle properties. This paper reports preliminary results on the viability of laser holography to the study of benthic sediment transport and particle visualization and builds upon earlier preliminary work on the holographic visualization of marine plankton (14, 15).
Materials and Methods Laser Type and Configuration. There are two main holographic arrangements that are potentially suitable for holography of sediments, viz., in-line (objects in transmitted light) and off-axis (objects in reflected light). However, inline holography provides several advantages over the offaxis method including highest resolution and minimum energy requirements of the laser. Under optimized recording and replay conditions, particle image resolution of 2-5 µm is achievable. The process, however, relies on transmission of the incident wave front through the scene, and this sets practical limitations on the upper size and concentration of the particles that can be recorded. As the size and concentration of the recorded objects increase, the transmitted reference beam is reduced, thereby reducing the overall signal-to-noise ratio (SNR) of the hologram (16). At increased concentrations, a larger number of particles are encompassed along the beam path, which increases the speckle noise and seriously affects the fringe contrast of holograms. Good holograms are usually achieved when the overall beam transmission is greater than about 80-90%. This corresponds to particle concentrations up to around 2 × 106 L-1 for particles around of 5 µm (17). Following the work of Meng and Hussain (18) and assuming an appropriate value of the SNR (ca. 10), equivalent number concentrations for particles of 20 and 100 µm are 150 000 and 25 000 particles/L, respectively. The upper limit to the recordable size is dependent on the recording wavelength and the distance between subject and film. A practical limit to the upper dimension is about 200-500 µm for objects close to the film (17). Thus, for higher concentrations of objects, off-axis holography is usually more suitable. Off-axis holograms have a practical lower limit of VOL. 35, NO. 11, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. (a) Recording system used with the erosion jet (CSM) and ruby laser. M1 and M2, 100% reflection (in certain conditions) mirrors for pulsed ruby. L0 and L2, concave lenses with different focal lengths. A1 and A2, apertures that limit the diameter of the beam. SF, spatial filter. L1, positive lens with long focal length. L3, collimator. (b) Replay system for image analysis. resolution of around 100 µm, which is also contingent upon the recording conditions, but the coherence length of the laser and available power only restrict the upper limit. The work reported here uses the in-line geometry. The majority of the holograms were recorded using the arrangement shown in Figure 1a. A Q-switched ruby laser emitting 30 mJ of energy in a single 50-ns pulse at a wavelength of 694.3 nm (red) was used. The laser was tuned to operate in single longitudinal (providing a coherence length of about 1 m) and transverse cavity modes (Gaussian beam profile). The beam was further “cleaned” by focusing through a spatial pinhole filter to remove unwanted noise and to ensure that the target was smoothly and cleanly illuminated. The beam was collimated before passing through the test tank. The hologram (the interference fringe pattern) is formed between the light directly transmitted through the water tank and the object beam diffracted by the sediment. The holograms are recorded on high-resolution holographic plates or film sensitized to the waveband being recorded (Agfa 8E75 for red). A stack of neutral density filters (0.3 or 0.6 D) were used, if needed, to reduce the overall light intensity such that the recorded optical density (OD) of the film is around 0.8-1.2 OD, corresponding to an exposure of between 44 and 88 µJ cm-2. This density level is known to produce good in-line holograms with high signal-to-noise. The exposure level was monitored using an integrated photodetector connected to an oscilloscope. An essential part of the holographic method is the replay of the holograms and data extraction. The configuration and 2276
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the optical components used in replay system are shown in Figure 1b. The incident beam is spatially filtered and collimated before illuminating the hologram. Two images are produced: a virtual image is located between the hologram and the illuminating laser; the second image, the real image, is formed between the hologram and the observer. It is this real image that forms the basis of data extraction from holograms. A video camera or measuring microscope can be traversed through the real image to produce “optical sectioning” of the recorded scene. The holograms were replayed using an argon laser providing a continuous beam at a wavelength of 514 nm. Analysis of the image was carried out using a CCD camera with or without objective lens as appropriate (this allowed a change of image magnification). The camera was positioned on a 3-axis, automatically controlled micro-positioning mounting and can be focused on any individual particle image in any plane. This permitted detailed positional and dimensional analysis to be carried out on the subject volume. Images were transferred to a computer and analyzed in detail using the image-processing software Zipshot. When recording particle dimensions, no correction was applied for image magnification, and the sizes reported are calibrated by using images of calibration wires of known dimension. Sediment Erosion. The cohesive strength meter (CSM) of Paterson (19) as modified by Tolhurst et al. (20) was used to induce erosion of sediments. The CSM employs a computer-controlled, pulsed, vertical axisymmetric water jet to erode sediments. The jet nozzle water pressure (P, kPa) was calibrated in terms of an equivalent horizontal shear
stress (τ0, Nm-2) relating to grain suspension (not initial motion) using noncohesive sand and spherical glass beads of known size (20):
τ0 ) 67(1 - e-P/310) - 195(1 - e-P/1623)
(1)
We recognize that the CSM device does not offer direct dynamic similarity to the detailed interfacial hydrodynamics characteristic of natural geophysical flows. However, the purpose of this work was simply to induce the suspension of particles in a controlled fashion in a laboratory setting. Experimental Methodology. Preliminary static experiments were first undertaken to explore the efficacy of particle visualization using in-line holography. Holograms were recorded under various conditions of particle concentration and sand particle size (250-500 µm). Grains were introduced into the laser beam by dropping them directly into the seawater tank. Additional experiments were then performed using the CSM on sand sediments that had been treated with a commercially available extracellular polysaccharide (EPS) from the bacterium Xanthomonas campestris (xanthan gum). Xanthan gum is used industrially as a plasticizer and emulsifyer in foodstuffs and also as a component in oil drilling operations (21), but in this context it mimics adhesive polymeric substances secreted by natural populations of bacteria and photosynthetic microalgae on intertidal mudflats (22-24). Both xanthan gum and natural EPS act as a “glue” by binding mineral grains together. Varying amounts of freeze-dried xanthan gum were dissolved in seawater (S ∼ 35, µ ∼ 1.072 × 10-3 Pa s-1) and then mixed thoroughly with wet beach sand to provide the following concentrations: 0 (control), 1.25, 2.5, and 5.0 g kg-1. These sediments were then poured into small Petri dishes and carefully submerged in the tank. Preliminary visual tests were made to assess the stress at which grains were first lifted into suspension. During actual erosion tests, the water jet and the laser were fired consecutively with approximately 0.5-s delay. Image Analysis. Three replicate holograms were collected for each EPS concentration. One hundred particle measurements were made from each hologram, and measurement of the long axis of individual grains or grain aggregates was aided by calibration wires of known thickness suspended vertically and horizontally in the tank. Four morphological particle types were recognizable in hologram images. These were as follows: (i) EPS-free particles, (ii) particles with attached EPS, (iii) aggregates of particles, and (iv) discrete EPS strands. The frequency of occurrence of each particle type was determined for the suite of holograms.
Results and Discussion Still Water In-Line Holography. Initial feasibility recordings using in-line holography of several tens of sand particles (fine-medium sand, median diameter ca. 214 µm) dropped directly into the tank provided encouraging results, and the sinking particles were easily resolved (Figure 2). The outline of grains was obvious, and microscale irregularities in the surfaces of some particles due to abrasion and weathering could be seen. More detail was discernible from the original hologram plates. Influence of EPS on Sediment Stability and Grain Morphology. Sediment Stability. The equivalent horizontal shear stresses necessary to suspend sand particles with increasing EPS concentration are given in Table 1. Generally there was a positive co-variation, and sediments with higher concentrations of EPS were more difficult to resuspend. The influence of EPS concentration was relatively linear for EPS concentrations up to 2.5 g kg-1, and the critical shear stress for suspension increased in a regular stepwise fashion within increasing EPS concentration. However, sediment dosed with
FIGURE 2. In-line two-dimensional representation of the hologram of settling medium-coarse sand particles (250-500 µm in diameter). Eleven sand grains are visible in a common focal plane. Surface irregularities are discernible for some grains. Out-of-focus particles correspond to particles at locations either less than or greater than camera focal length and may be brought into focus by adjusting the position of the camera (scale bar 200 µm).
TABLE 1. Critical Erosion Stress (as kPa and Nm-2) for Suspension of Sand versus Concentration of EPS (Extracellular Polymeric Substance) control
[EPS] (g kg-1)
sand (kPa)
τocr (Nm-2)
0.00 1.25 2.50 5.00
17.2 27.6 44.8 227.5
1.61 2.58 4.15 19.30
5 g kg-1 were particularly stable (τ0cr )19.30 Nm-2), and benthic currents in excess of about 0.4 ms-1 would be necessary to transport these sediments in the natural environment. A positive co-variability between τ0cr and EPS concentration has been observed elsewhere for both artificial and natural sediments (e.g., refs 22, 25, and 26). Tolhurst (27), for instance, reported a 3-fold linear increase in τ0cr and a concomitant decrease in erosion rate for xanthan gum concentrations up to 10 g kg-1 on cleaned fine-grained mud from the Eden Estuary. Specialized (low-temperature) scanning electron microscopy (L-TSEM) reveals the mechanism through which EPS exerts a control over intergrain contacts and modifies stability for noncohesive sands (Figure 3). L-TSEM is particularly suited to the visualization of hydrated biopolymers (e.g., refs 28 and 29), since quench-freezing using liquid nitrogen does not disrupt sediment fabric on a micrometer scale (30), and there is no necessity for the replacement of cellular/ extracellular water. Figure 3a shows the tertiary colloidal structure of EPS in seawater (no mineral grains). Decho (24) describes this type of structure as intermediate in state between truly dissolved organic material and condensed, tightly wound matrix gels. The EPS exists as a tangible and extensive fibrillar network rather than as a contiguous sheet, although a significant degree of microscale heterogeneity is observed and thin strands are often co-located with denser, thicker regions (to the left on the image). Decho (24) notes that mucilages such a xanthan gum typically display commensurate microscale and molecular scale variability in cohesiveness where generally the condensed regions are more cohesive than looser conformations. This may prove of importance to our studies as it may govern where the matrix (which is a weak viscoelastic gel with relatively few lateral branches from the glucose backbone) tears when exposed VOL. 35, NO. 11, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Low-temperature scanning electron microscope images of (a) partially freeze-dried EPS “dissolved” in seawater (note the natural fibrillar network structure (scale bar 100 µm)); (b) EPS and sand treated with 1.25 g kg-1 EPS; low magnification image showing strands and threads of EPS (note the continuous swath of EPS down the right-hand margin (scale bar 1000 µm)); and (c) high magnification view detailing polymer bridging between grains as well as interstitial EPS (scale bar 100 µm); (d) low magnification image at EPS concentration of 5 g kg-1 showing generally greater occlusion of pores by polymer (scale bar 1000 µm). to turbulent fluid stress. This will be reflected in the morphologies of resuspended grain aggregates. At low concentrations (1.25 g kg-1), EPS is seen to bridge pore throats with interconnecting strands and sheets of elastic polymer rather than envelop grains completely (Figure 3b,c). The presence of the cohesive, interparticle polymer bridges augments the gravitational and frictional interlocking forces that act to retain grains on the bed, and consequently, greater fluid drag is necessary to lift grains from the sediment. The sediment is dramatically stabilized through addition of 5 g kg-1 EPS (Table 1), and electron microscopy reveals a more occluded pore structure as well as more numerous intergrain strands and linkages (Figure 3d). However, mineral grains are not completely smothered by EPS at this concentration (or higher, e.g., 20 g kg-1; unpublished data). Other observations (e.g., refs 31-33) suggest that saturation of mineral surfaces by organic monolayers is more typical of finergrained sediments. Sand particles, because of their greater size, are only partially smothered in EPS at concentrations up to 5 g kg-1. Nonetheless, the experimental EPS concentrations are similar to concentrations found in natural intertidal sediments (e.g., refs 32 and 33), thus the stabilization effect and the interparticle bridging observed in these experiments may mirror processes in the field situation. Particle Morphology. Figure 4 shows a montage of holographic images of clean sand grains (control) suspended using the CSM jet. A hologram of particle-free seawater is provided for comparison. Individual grains of a variety of shapes and sizes were lifted into suspension by the turbu2278
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lence. Each grain responded to the fluid turbulence as a discrete particle not influenced by neighboring grains. A mean grain diameter (dmean) from replicate control holograms is 172 µm (n ) 275), and the respective smallest (dmin) and largest (dmax) grain diameters are 37 and 430 µm. This compares favorably with that obtained from Fraunhofer (laser) particle size analysis (dmean ) 218 µm; dmin ) 106 µm; dmax ) 420 µm), although the holographic images show that some coarse silt grains (30-62.5 µm) are present within the sample. Sand grains across the entire spectrum of sizes are thus resuspended by the jet (τ0cr ) 2.5 Nm-2), which is to be expected on the basis of the instrument calibration (20, Table 1). Aggregation of resuspended sand particles was common after EPS addition (Figure 5, EPS at 1.25 g kg-1). Highly reflective material was associated with some aggregates (Figure 5), and this is believed to be the xanthum polymer. This conclusion is supported by numerous observations of unattached but highly reflective entities only evident in sediments treated with EPS and by holograms of seawater containing only colloidal EPS (Figure 6). Increases in EPS concentration to 2.5 g kg-1 produced large grain aggregates, although many clean individual grains were also obvious (Figure 5). This supports the earlier contention that the EPS did not attach uniformly to all grains (see Figure 3d). Detailed inspection of single-grain EPS aggregates showed that the EPS was not in the form of a confluent monolayer but appears patchy on the surface of the grain or as “clumps” of EPS associated with only a small portion of the grain surface
FIGURE 4. Holograms of sand grains resuspended by the CSM jet: (a) montage of sand grains for comparison with (b) hologram of seawater only (scale bar 100 µm).
TABLE 2. Number of Specified Particle Types (total n ) 300) Found in Suspension Following Erosion versus Increasing EPS (Xanthan Gum) Concentration [EPS] (g kg-1)
no. of EPS particles
no. of free grains
no. of grain aggregates
no. of sand + EPS aggregates
control 1.25 2.5 5
0 56 80 161
298 230 204 111
2 7 8 4
0 7 8 24
(Figure 5c). This essentially reflects the microphysical association of EPS with mineral grains in the original (preerosion) sediment bed. Aggregate morphologies were substantially different from the roughly spherical shape of free sands. Aggregates were typically nonspherical and angular in form, similar morphologically to natural estuarine aggregates (e.g., ref 34). Replicate holographic images for each treatment were analyzed to determine specific particle types: the number of discrete EPS particles, the number of clean sand particles, the number of multigrain aggregates, and the number of sand-EPS particles (Table 2). This approach confirms the broad qualitative interpretation that addition of EPS to clean sand increases the abundance of both grain-grain aggregates bound together by EPS and single grains associated with EPS flakes. The number of discrete EPS threads, presumably arising from polymer detachment from grain surfaces under instantaneously high shear rates else actual tearing of the
FIGURE 5. Influence of EPS on the aggregation of sand particles. (a) Real hologram of sand treated with 1.25 g kg-1 EPS; encircled areas show grains bridged by EPS. (b) Montage of sand treated with 2.5 g kg-1 EPS; note the large aggregate smothered by EPS (AGG) and the single strand of EPS (EPS). (c) Montage of sand treated with 5 g kg-1 EPS; note attachment of EPS on the periphery of grains (PER) and large attached clumps (CLU) and free floating EPS (EPS). In panel a, the scale bar is 200 µm; in panels b and c, the scale bars are 100 µm. polymer strands, also follows this trend. The proportion of clean sand grains varies inversely with EPS concentration. Application of the χ2 test after aggregating the replicate observations for each EPS treatment (Table 2) confirms that the distribution of particle types relating to each of the EPS treatments were significantly different (χ29 ) 301.84; R ) 0.05). VOL. 35, NO. 11, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 6. Hologram image of dissolved/colloidal EPS only (center scale bar 100 µm). Environmental Significance of EPS. Bennett et al. (35) describe polymer bridging of sediment particles as a biophysical mechanism through which microorganisms can modify particle properties. Changes to the physical properties of sediment particles can potentially have a major affect on the dynamic cycling of sediments both between the bed and the overlying water column and within the wider sedimentary system. This study and others (22, 25-27) have shown clearly the dramatic effect even small quantities of EPS can have on the critical suspension threshold, τ0cr (Table 1). Viewed within a tidal framework, increases in τ0cr will reduce the proportion of the tidal cycle under which resuspension may occur and consequently reduce the overall volume of sediment scoured. Kornmann and de Deckere (36) provide a clear example of this from the Ems-Dollard Estuary. In addition, bio-aggregation increases the particle diameter (e.g., Figure 5), and this will give rise to an enhanced settling rate as well as reducing net horizontal transport. On the other hand, organic material like EPS is of very low density (approximately that of seawater); therefore, incorporation of EPS within aggregates may act to reduce settling rate. Indeed, thick adhesive layers of EPS found within well-developed biofilms are known to be buoyant (37) and promote erosion. In addition to forming a component of bacterial and microalgal exudates, EPS is also an ideal food source for many heterotrophic microorganisms, and aggregates provide a surface for bacterial attachment and growth. Suspended aggregates in estuarine systems commonly have very high rates of heterotrophic metabolism (38) and form an important link in detrital carbon transfer through both pelagic and benthic environments. EPS may thus be a key component in marine ecosystems from both a physical and a biological perspective. Clearly, the consequences of biophysical influence on marine sediments can be marked, but the net influence in natural environments is complex and, as yet, not amenable to either modeling or prediction. Experimental Limitations. The pulsed jet experimental system was used to induce turbulent bursts at the sediment bed. While this may actually mimic natural erosion in some cases (39, 40), in this work we have not attempted to mimic natural systems but rather to induce incipient erosion without the complication of further particle modification through high, post-erosion turbulent intensities. The influence of flow and turbulence after erosion will form the basis of future studies now that the system has been validated. This will be achieved using the erosion chamber of Gust and Muller (39), which exerts a tangential fluid stress on surfaces and offers direct dynamic similarity to the detailed interfacial hydrodynamics characteristic of natural geophysical flows (e.g., 2280
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the sea). Furthermore, technology is advancing rapidly, and recently a combined off-axis/in-line “holo-camera” has been deployed successfully from a ship in a Scottish sea loch (Watson, unpublished data). Hence, the possibility exists for translation of our experiments to natural marine environments. Xanthan gum is a natural bacterial product (23, 24) and as such is a reasonable analogue for natural EPS. The fibrillar network structure of xanthan gum observed in our studies (e.g., Figure 3a) is morphologically similar to that associated with microbes in intertidal environments (e.g., refs 11, 12, and 28). However, the spectrum of biopolymers in nature will be far greater than under our controlled experiments, and the microscale distribution within natural sediments is probably much more heterogeneous. The effects of both these factors will require further study. The cohesiveness and microphysical properties of biopolymers may also change according to environmental conditions, such as water content (degree of soil hydration) and temperature. Ultimately, therefore, experiments will be conducted using natural sediments from a variety of coastal sedimentary environments (e.g., intertidal mudflat, mixed flat, biogenic, and quartz beach sands) under a series of differing environmental conditions.
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(24) Decho, A. In Biostabilisation of Sediments; Krumbein, W., Paterson, D. M., Stal, L., Eds.; BIS: Oldenburg, Germany, 1994; pp 135-148. (25) Grant, J.; Bathmann, U. V.; Mills, E. L. Estuarine Coastal Shelf Sci. 1986, 23, 225-238. (26) Black, K. S. In Cohesive Sediments; Burt, et al., Eds.; Wallingford, England, 1997; pp 231-244. (27) Tolhurst T. J. Microbial mediation of intertidal sediment stability. Ph.D. Thesis (unpublished), St. Andrews University, 1999. (28) Paterson, D. M. J. Geol. Soc., London 1995, 152, 131-140. (29) Chenu, C.; Jaunet, A. M. Scanning 1992, 14, 360-364. (30) Wiltshire, K. H., Blackburn, J.; Paterson, D. M. J. Sediment. Res. 1997, 67, 977-981. (31) Taylor, I.; Paterson, D. M. Estuarine Coastal Shelf Sci. 1998, 46, 359-370. (32) Taylor, I.; Paterson, D. M.; Mehlert, A. Biogeochemistry 1999, 45, 303-327. (33) Weiler, R. R.; Mills, A. A. Deep Sea Res. 1965, 12, 511-529. (34) Van Leussen, W. In Physical Processses in Estuaries; Dronkers, J., van Leussen, W., Eds.; Springer: Berlin, 1998; pp 347-403.
(35) Bennett R. H., O’Brien, N. R., Hulbert, M. H., Eds. Microstructure of Fine-grained Sediments; Frontiers in Sedimentary Geology; Springer-Verlag: Berlin, 1990; pp 73-92. (36) Kornmann, B. A.; de Dekere, E. M. G. T. J. Geol. Soc. London 1998, 139, 231-242. (37) Sutherland, T. F.; Grant J.; Amos C. L. Limnol. Oceanogr. 1998, 43 (1), 65-72. (38) Kepkay, P. E.; Schwinghammer, P.; Willar, T.; Bowen, A. J. Appl. Environ. Microbiol. 1986, 51, 163-170. (39) Gust, G.; Muller, V. In Cohesive Sediments; Burt, et al., Eds.; Wallingford, England, 1997; Chapter 10. (40) Ruddy, G. R.; Turley, C. M.; Jones, T. E. R. J. Geol. Soc. London 1998, 139, 135-148.
Received for review July 11, 2000. Revised manuscript received March 12, 2001. Accepted March 14, 2001. ES0014739
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