We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the ␤-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of ␤-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the ␤-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.

The objective of this study was to investigate the autoaggregation, coaggregation and biofilm formation of four bacteria namely Sphingobium, Xenophilus, Methylobacterium and Rhodococcus isolated from drinking water. Auto and coaggregation studies were performed by both qualitative (DAPI staining) and semi-quantitative (visual coaggregation) methods and biofilms produced by either pure or dual-cultures were quantified by crystal violet method. Results from the semi-quantitative visual aggregation method did not show any immediate auto or coaggregation, which was confirmed by the 4′,6 diamidino-2-phenylindole (DAPI) staining method. However, after 2 hours, Methylobacterium showed the highest autoaggregation of all four isolates. The Methylobacterium combinations showed highest coaggregation between dual species at extended period of times (72 hours). Biofilm formation by pure cultures was negligible in comparison to the quantity of biofilm produced by dual-species biofilms. The overall results show that coaggregation of bacteria isolated from drinking water was mediated by species-specific and time-dependent interactions with a synergistic type of biofilm formation. The results of this study are therefore a useful step in assisting the development of potential control strategies by identifying specific bacteria that promote aggregation or biofilm formation in drinking water distribution systems.

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Aims:  To determine the spatial and temporal variability in the abundance, structure and composition of planktonic bacterial assemblages sampled from a small, looped water distribution system and to interpret results with respect to hydraulic conditions. Methods and Results:  Water samples were collected from five sampling points, twice a day at 06:00 h and 09:00 h on a Monday (following low weekend demand) and a Wednesday (higher midweek demand). All samples were fully compliant with current regulated parameter standards. This study did not show obvious changes in bacterial abundance (DAPI count) or community structure Denaturing gradient gel electrophoresis analysis with respect to sample site and hence to water age; however, the study did show temporal variability with respect to both sampling day and sample times. Conclusions:  Data suggests that variations in the bacterial assemblages may be associated with the local system hydraulics: the bacterial composition and numbers, over short durations, are governed by the interaction of the bulk water and the biofilm influenced by the hydraulic conditions. Significance and Impact of the Study:  This study demonstrates general stability in bacterial abundance, community structure and composition within the system studied. Trends and patterns supporting the transfer of idealized understanding to the real world were evident. Ultimately, such work will help to safeguard potable water quality, fundamental to public health.

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The biogeochemical cycles of elements from soils to plants are mainly governed by their rhizosphere processes. Understanding these processes is challenging and remains largely unresolved due to the complex interrelationships among different elements and due to a lack of appropriate techniques for simultaneous spatiotemporal monitoring. MethodsThis study employed an In-situ Porewater Iterative (IPI) sampler array (0-22 mm measurement distance every 1.7 mm, with a time interval of 3 to 10 days) to capture the in situ spatiotemporal dynamics of ten elements (Fe, Mn, As, P, S, Cr, Co, Zn, Sb and Cd) in the paddy rhizosphere to examine their covarying changes in time and space dimensions. ResultsThe ndings revealed that the solute-phase concentration of most elements, other than Sb and Cd, increased to a peak after 30 days of paddy soil ooding and then decreased. Additionally, Sb and Cd continuously decreased during ooding. Fe (-52%), Mn (-17%), P (-43%), Co (-11%), and As species (-74%) were substantially immobilized within a 10 mm zone around the roots, while Zn (28%) and Cd (41%) increased. The greater immobilization of As and re-mobilization of Cd, in the rhizosphere, are stimulated by biotic oxidation of arsenite to arsenate with root oxygen loss and the pH decrease, respectively. ConclusionsOur study showed most sampled elements covaried with Fe both in time and space in the rhizosphere, but the elements are temporally and spatially determined by multiple biogeochemical processes in soils as well as exudates from plant roots.

The aim of the study was to investigate the acclimation of precultivated acetate-fed aerobic granular sludge to a toxic xenobiotic biodegradation. Establishment of p-nitrophenol (PNP) biodegradation in acetate-fed aerobic granular sludge and concomitant changes in the microstructure and bacterial community were determined. Rapid establishment of PNP utilization was observed in the granular sludge when fed with PNP as the sole carbon source. The specific PNP removal was 36-mg h(-1) g(-1) granular biomass at an initial PNP concentration of 50 mg L(-1). The presence of PNP resulted in significant membrane damage in a subpopulation of the bacterial consortium, as shown by BacLight viability staining. This was coincided with a significant decrease in the culturable bacterial diversity of the granular biomass. PCR-DGGE analysis revealed a shift and decrease in number of bands during the establishment of PNP biodegradation. Scanning electron microscopy showed the dominance of rod-shaped bacteria in the PNP-utilizing microbial granules. Our results suggest that acetate-fed granular sludge could be quickly adapted for PNP biodegradation.

Terrestrial leaf-litter (LL) inputs impose great bottom-up effects on freshwater ecosystems by fueling detritus-based food webs, affecting macroinvertebrate and microbial communities, and influencing ecosystem functioning. However, increasing intensive anthropogenic activities including the inputs of herbicide glyphosate disturb the breakdown of LL in streams. In this study, an anthropogenic carrion subsidy (chicken meat) and glyphosate (a stressor) were used to investigate their individual and combined effects on LL breakdown in urban streams and forest streams in China. We found that: 1) carrion subsidy decreased LL breakdown rate in both urban and forest streams and increased total and predator richness in forest streams, the reduced LL breakdown rates may be attributed to the foraging shift of macroinvertebrates from LL to carrion subsidy; 2) glyphosate depressed LL breakdown rate in forest but not in urban streams, the reduced LL breakdown rate may be caused by the negative effects on microbes; 3) forest streams showed significantly higher LL breakdown rates in both coarse and fine mesh bags than urban streams which were induced by the high dissolved oxygen (DO) and collector-gatherer richness. Our results provide evidence that LL breakdown in streams is sensitive to inputs of anthropogenic carrion subsidy and glyphosate through the impacts on macroinvertebrates and microbes, respectively. Furthermore, this study underscores the importance of local macroinvertebrate and microbial communities when assessing the responses of stream ecosystem functioning and macroinvertebrate communities to multiple stressors, as the individual and combined effects of stressors can be site-specific in streams with different physical characteristics and biological communities.