Stable carbon isotope ratios of bacterial biomarkers were determined to infer sources of organic carbon used by bacteria in the sediments of three salt marshes. Biomarkers studied were polar lipid-derived fatty acids (PLFA), mainly bacteria-specific, methyl-branched i15 : 0 and a15 : 0. Experiments showed that isotopic fractionation between substrate and biomarkers was relatively constant (Ϫ4 to Ϫ6‰, on average) compared to the wide range in 13 C/ 12 C ratios of carbon sources found in the studied marshes. At the Spartina site of the Waarde Marsh (The Netherlands), biomarker 13 C/ 12 C ratios were depleted by approximately 6‰ more than expected for bacteria growing on Spartina litter and were similar to an unvegetated control sediment. This pattern suggested that local macrophyte production was of little importance and that other material (probably of algal origin) was the dominant carbon source for bacterial growth. Spartina contributed about half of the carbon in bacterial PLFA at the Kattendijke Marsh (The Netherlands) and dominated at the Great Marshes (U.S.). The variation in bacterial carbon sources in these marshes was probably related to estimated inputs of nonmacrophyte organic matter to the sediment. At the Waarde Marsh, a clear plant species effect was found as coupling between plant and bacteria was more important in Scirpus maritimus than in Spartina anglica. The contribution of local plant production to bacterial biomass in salt-marsh sediments is highly variable between marshes and depends on the input of nonmacrophyte material by sedimentation in comparison to local plant input, which in turn may differ among plant species.

Two field studies were conducted to study the in situ net production of extracellular carbohydrates and the distribution of benthic diatoms over a period of 24 h per study. A comparison was made between a situation where a clear surface biofilm of diatoms had developed and a situation where this was not the case. Vertical profiles were made by sampling the top 2 mm of the sediment at depth intervals of 0.2 mm using the 'cryolander' technique. In the presence of a biofilm, diatom distribution showed a consistent pattern when the sediment was emersed. In the light, most of the diatoms were present in the top 0.2 mm while in the dark, diatoms were homogeneously distributed in the upper 2 mm of the sediment. When a biofilm was absent, no clear patterns were observed. Extracellular carbohydrates were extracted from the sediment and separated in 2 operationally defined fractions (colloidal and EDTA-extractable). The 2 carbohydrate fractions showed a different dynamic behaviour. The colloidal carbohydrate fraction was highly variable while the EDTAextractable fraction behaved more conservatively. Only in the light and in the presence of a diatom biofilm, was production of extracellular carbohydrates observed. The maximum rate of chlorophyllnormalized production of extracellular carbohydrates, expressed in glucose equivalents (g g -1), amounted to 20 h -1 in the upper 0.2 mm. The molecular size distribution of both carbohydrate fractions was similar. The monosaccharide composition was also similar, except that the EDTAextractable fraction contained a higher percentage of uronic acids. Carbohydrates produced during tidal emersion were rich in glucose and were rapidly turned over.

BackgroundConfocal laser scanning microscopy (CLSM) is the method of choice to study interfacial biofilms and acquires time-resolved three-dimensional data of the biofilm structure. CLSM can be used in a multi-channel modus where the different channels map individual biofilm components. This communication presents a novel image quantification tool, PHLIP, for the quantitative analysis of large amounts of multichannel CLSM data in an automated way. PHLIP can be freely downloaded from ResultsPHLIP is an open source public license Matlab toolbox that includes functions for CLSM imaging data handling and ten image analysis operations describing various aspects of biofilm morphology. The use of PHLIP is here demonstrated by a study of the development of a natural marine phototrophic biofilm. It is shown how the examination of the individual biofilm components using the multi-channel capability of PHLIP allowed the description of the dynamic spatial and temporal separation of diatoms, bacteria and organic and inorganic matter during the shift from a bacteria-dominated to a diatom-dominated phototrophic biofilm. Reflection images and weight measurements complementing the PHLIP analyses suggest that a large part of the biofilm mass consisted of inorganic mineral material.ConclusionThe presented case study reveals new insight into the temporal development of a phototrophic biofilm where multi-channel imaging allowed to parallel monitor the dynamics of the individual biofilm components over time. This application of PHLIP presents the power of biofilm image analysis by multi-channel CLSM software and demonstrates the importance of PHLIP for the scientific community as a flexible and extendable image analysis platform for automated image processing.

The sediment-stabilizing effect of benthic diatoms was investigated in a laboratory setting. Axenic cultures of the benthic diatoms Nitzschia cf. brevissima and Cylindrotheca closterium were inoculated in Petri dishes containing sand and incubated under axenic conditions. By ensuring aseptic routines throughout the experiments, interference from other organisms occurring with diatoms in natural photothrophic biofilms was avoided. This allowed the examination of the role of benthic diatoms in sediment stabilization. Increases in the critical erosion shear stress of the sediment were observed in the presence of both diatom taxa relative to sterile sediment. However, N. cf. brevissima was more effective than C. closterium. Values of critical shear stress in the experimental system were in the same range as those observed in natural biofilms, which indicates that diatoms are important agents for biogenic stabilization. Extracellular carbohydrate contents in the microcosms were similar for both diatom species. However, in the presence of N cf. brevissima, extracellular carbohydrate correlated significantly to critical shear stress, explaining up to 80% of the variation, whereas this was not the case for C. closterium. Therefore, it was concluded that the quantity of extracellular polymeric substances (EPS) alone did not explain the biogenic stabilization. Observed adsorption of EPS to sediment particles depended on the relative amount of uronic acids in the exopolymers. Using fluorescently labeled lectins, confocal laser scanning microscopy showed that EPS secretion by N. cf. brevissima resulted in ordered three-dimensional matrix structures. It is suggested that the structuring of EPS plays an prominent role in the process of biostabilization, and that diatoms such as N. cf. brevissima are actively involved in producing the structure of EPS, whereas others such as C. closterium do not do so to the same extent.

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