Marine mesocosms were manipulated with inorganic nutrients over a period of 22 d to investigate organic carbon partitioning under a variety of nutrient regimes. The chemical analyses and biotic measurements included inorganic nutrients, pigment signatures, particulate and dissolved organic species, bacterial production, and community respiration. The biodegradability of dissolved organic carbon (DOC) was investigated with in vitro decomposition experiments.The net particulate organic carbon (POC) production was 50% of the total organic production during the initial 6 d of nutrient-replete growth and during a major diatom bloom. In all other situations the carbon partitioning was strongly in favor of DOC, which accounted for 82 to 111% of the total production. The production of new DOC preceded new DON by about 1 week. Thus, the new dissolved organic matter (DOM) initially had an infinite C : N ratio, which fell to 11-20 when DON started to accumulate. The highest C : N ratio was measured during a nutrientreplete diatom bloom. Dissolved polysaccharides accounted for 50 to 70% of the new DOC, and the lowest relative amount was produced during a diatom bloom. The chemical analyses unequivocally demonstrated that carbon partitioning in favor of carbon-rich DOM can take place during an active diatom bloom and not only during the decay of a bloom. The DOC-producing mechanisms cannot be fully identified. However, during the different growth phases the DOC production varied, as did the speciation of DOM with respect to the C : N ratios. When net 1 Corresponding author (MSondergaard@zi.ku.dk).

Aims:  To determine if orally ingested Bacillus spores used as probiotics or direct‐fed microbial feed additives germinate and the vegetative cells grow in the gastrointestinal (GI) tract. Methods and Results:  Three independent experiments were done to determine if spores of Bacillus licheniformis and Bacillus subtilis germinate and grow in the GI tract of pigs. After a 2 weeks spore‐feeding period, spores were detected in all segments of the GI tract. The lowest number of spores was found in the stomach, increasing in the small intestine to approx. 55% of the dietary inclusion. When spores were withdrawn from the feed, faecal excretion of spores reflected the dietary inclusion, but decreased gradually to the background level after 1 week. By containing spores in short, sealed pieces of dialysis membrane that were orally administered to the pigs, both the number of spores and vegetative cells could be determined by flow cytometry. Spores accounted for 72% of the total counts after 4–6 h in the stomach and proximal part of the small intestine. After 24 h, spores constituted only 12% of the total counts in the stomach, caecum, and mid‐colon. Less spores and more vegetative cells were detected after 24 h, but total counts increased only 2·14‐fold compared to time zero. Conclusions:  The experiments showed that 70–90% of dietary‐supplemented Bacillus spores germinate in the proximal part of the pig GI tract, and that only limited outgrowth of the vegetative cell population occurs. The two Bacillus strains can temporarily remain in the GI system, but will be unable to permanently colonize the GI tract. Significance and Impact of the Study:  A substantial population of growing vegetative cells in the GI tract is not a prerequisite for the mode of action of Bacillus feed additives and probiotics.

ABSTRACT. We studied the ecology of heterotrophic bacteria attached to the mucilaginous colonies of Microcystis spp. (Cyanobacteria) in the eutrophic lake Frederiksborg Slotsse, Denmark. The succession in bacterial abundance, production and potential aminopeptidase activity in 20 pm fractionated samples was followed during periods in which Microcystis dominated the phytoplankton. We operationally defined that nets of 20 pm mesh-width segregated bacteria associated with Mjcrocystis (Microcystisassociated bacteria, MB; s 2 0 pm size fraction) from the mainly free-living bacteria in the filtrate (FB, <20 pm size fraction). According to this definition, the contribution of MB during summer 1995 and autumn 1994, respectively, averaged 10 i 4 (i standard deviation) and 37 i 1 2 % of total bacterial biomass and 25 i 13 and 43 i 16% of total bacterial production, as estimated from thymidine (TdR) incorporation. During summer, MB further contributed 55 i 18% of total leucine incorporation measured at 600 nM leucine and 53 i 12% of total potential aminopeptidase activity. Although 20 pm mesh-width nets also retained particles other than Microcystis, our results indicate that Microcystis was a 'hotspot' for bacterial activity, comparable to larger aggregates known as marine or lake snow. During summer, growth rate and specific aminopeptidase activity of MB generally exceeded those of FB, which points to diversified microenvironments or species compositions. In order to balance gain and loss rates within the community of MB, we hypothesize that a large fraction of MB produced were exported from Microcystis to the surrounding water, only modified by the loss due to viral lysis. This idea arose from reported low loss rates of Microcystis and continuous measures indicating that a surplus of more than 70% of MB production (TdR) was not reflected as biomass increases within the community of MB. According to this hypothesis Microcystis may be considered as a bacterial 'incubator' for the surrounding water.

Recent studies employing reporter gene technology indicate that the availabilities of the major nutrients nitrogen, phosphate, and iron to Pseudomonas are not severely limited in bulk soil. Indirect evidence has pointed to carbon limitation as a severe nutritional stress in this environment. We show that a plasmid (pGM115)-borne transcriptional fusion between the S -dependent Escherichia coli promoter P fic and lacZ functions as a reliable reporter for carbon availability in Pseudomonas fluorescens. When P. fluorescens strain DF57 (pGM115) was introduced into bulk soil, carbon-limiting conditions were indicated by citrate-repressible induction of ␤-galactosidase activity. To address carbon availability at the single-cell level, we developed an immunofluorescence double-staining procedure for individual DF57 cells expressing ␤-galactosidase from P fic . Changes in cell size and expression of ␤-galactosidase were analyzed by flow cytometry. Cells extracted from soil microcosms reduced their size less than carbon-starved cells in pure culture and showed an increased tendency to aggregate. The single-cell analysis revealed that for cells residing in soil, the expression of ␤-galactosidase became heterogeneous and only a DF57 subpopulation appeared to be carbon limited. In soil amended with barley straw, limited nitrogen availability has been determined by use of the bioluminescent reporter strain P. fluorescens DF57-N3. We used strain DF57-N3(pGM115) as a double reporter for carbon and nitrogen limitation that allowed us to study the dynamics of carbon and nitrogen availabilities in more detail. In strawamended soil ␤-galactosidase activity remained low, while nitrogen limitation-dependent bioluminescence appeared after a few days. Hence, nitrogen became limited under conditions where carbon resources were not completely exhausted.

Phytoplankton blooms were created in freshwater enclosures to study the functional succession and diversification for attached (>10 µm size fraction) and free-living (<10 µm size fraction) assemblages of bacteria. Bacterial dynamics in abundance, production and enzyme activity was monitored by standard methods. The functional diversity with respect to sole carbon source utilization was assessed with Biolog GN plates inoculated with bacteria from the 2 size fractions. Moreover, bacterial isolates were screened for enzyme activity involved in the degradation of carbohydrates, chitin, protein and lipid. As the bloom proceeded, the functional diversity of attached and free-living bacteria appeared to be very similar. Most functional groups present in the free-living bacterial assemblage were also recovered attached to particulate matter. Additionally, the distribution of bacterial isolates with enzyme expression was similar with respect to 6 enzymes (p > 0.07), whereas 3 glucoside-bond cleaving enzymes were more frequent among the free-living isolates (p < 0.02). These results indicate that attached and free-living bacteria were functionally closely related and that their succession tended to converge during the phytoplankton bloom. Hence, bacteria attached to particulate matter are not necessarily functionally distinct and specialized for polymer hydrolysis relative to the free-living assemblage, though they are often anticipated to be different due to their close association with particulate polymers.