Biofilm Abundance Research Articles

Forestry affects the abundance of Phormidium-dominated biofilms and the functioning of a New Zealand river ecosystem

Toxic cyanobacterial proliferations in water bodies can cause serious environmental and public health issues, as well as having economic effects. Increased inputs of nutrients and fine sediment caused by forestry have been hypothesised as possible causes of increased Phormidium-dominated proliferations in New Zealand rivers. Little is known about the effect of these proliferations on river ecosystem functioning. In the present study, we evaluated five sites along the Maitai River (New Zealand) differing in pine plantation cover of their catchments. We hypothesised that Phormidium biofilms would trap more sediments and recycle more phosphorus than diatoms, that Phormidium proliferations would increase with forestry cover in the catchment and that the varying abundance of Phormidium would affect river ecosystem functioning. Phormidium did not trap more sediment or recycle more phosphorus (measured as alkaline phosphate activity) than diatom biofilms. However, the cover of Phormidium did increase with the proportion of forestry in the catchment. Organic matter decomposition rates (measured as loss of tensile strength of standard cotton strips) varied very little among sites, whereas river ecosystem metabolism increased with the abundance of Phormidium, especially in the lower part of the river. The results of the present study suggest that pine forestry does promote Phormidium biofilm abundance and affect ecosystem functioning in the Maitai River.

Comparison of microbial communities of activated sludge and membrane biofilm in 10 full-scale membrane bioreactors

Operation of membrane bioreactors (MBRs) for wastewater treatment is hampered by the membrane biofouling resulting from microbial activities. However, the knowledge of the microbial ecology of both biofilm and activated sludge in MBRs has not been sufficient. In this study, we scrutinized microbial communities of biofilm and activated sludge from 10 full-scale MBR plants. Overall, Flavobacterium, Dechloromonas and Nitrospira were abundant in order of abundance in biofilm, whereas Dechloromonas, Flavobacterium and Haliscomenobacter in activated sludge. Community structure was analyzed in either biofilm or activated sludge. Among MBRs, as expected, not only diversity of microbial community but also its composition was different from one another (p < 0.05). Between the biofilm and activated sludge, community composition made significant difference, but its diversity measures (i.e., alpha diversity, e.g., richness, diversity and evenness) did not (p > 0.05). Effects of ten environmental factors on community change were investigated using Spearman correlation. MLSS, HRT, F/M ratio and SADm explained the variation of microbial composition in the biofilm, whereas only MLSS did in the activated sludge. Microbial networks were constructed with the 10 environmental factors. The network results revealed that there were different topological characteristics between the biofilm and activated sludge networks, in which each of the 4 factors had different associations with microbial nodes. These results indicated that the different microbial associations were responsible for the variation of community composition between the biofilm and activated sludge.

Compositional Analysis of Biofilms Formed by Staphylococcus aureus Isolated from Food Sources.

Sixteen Staphylococcus aureus isolates originating from foods (eight from dairy products, five from fish and fish products and three from meat and meat products) were evaluated regarding their biofilms formation ability. Six strains (E2, E6, E8, E10, E16, and E23) distinguished as strong biofilm formers, either in standard Tryptic Soy Broth or in Tryptic Soy Broth supplemented with 0.4% glucose or with 4% NaCl. The composition of the biofilms formed by these S. aureus strains on polystyrene surfaces was first inferred using enzymatic and chemical treatments. Later on, biofilms were characterized by confocal laser scanning microscope (CLSM). Our experiments proved that protein-based matrices are of prime importance for the structure of biofilms formed by S. aureus strains isolated from food sources. These biofilm matrix compositions are similar to those put into evidence for coagulase negative staphylococci. This is a new finding having in view that scientific literature mentions exopolysaccharide abundance in biofilms produced by clinical isolates and food processing environment isolates of S. aureus.

Inocula selection in microbial fuel cells based on anodic biofilm abundance of Geobacter sulfurreducens

Microbial fuel cells (MFCs) rely on microbial conversion of organic substrates to electricity. The optimal performance depends on the establishment of a microbial community rich in electrogenic bacteria. Usually this microbial community is established from inoculation of the MFC anode chamber with naturally occurring mixed inocula. In this study, the electrochemical performance of MFCs and microbial community evolution were evaluated for three inocula including domestic wastewater (DW), lake sediment (LS) and biogas sludge (BS) with varying substrate loading (Lsub) and external resistance (Rext) on the MFC. The electrogenic bacterium Geobacter sulfurreducens was identified in all inocula and its abundance during MFC operation was positively linked to the MFC performance. The LS inoculated MFCs showed highest abundance (18%±1%) of G. sulfurreducens, maximum current density [Imax=(690±30)mA·m−2] and coulombic efficiency (CE=29%±1%) with acetate as the substrate. Imax and CE increased to (1780±30)mA·m−2 and 58%±1%, respectively, after decreasing the Rext from 1000Ω to 200Ω, which also correlated to a higher abundance of G. sulfurreducens (21%±0.7%) on the MFC anodic biofilm. The data obtained contribute to understanding the microbial community response to Lsub and Rext for optimizing electricity generation in MFCs.

Microalgal biofilms on common yew needles in relation to anthropogenic air pollution in urban Prague, Czech Republic

Excessive occurrence of microalgae on needles of gymnosperms was reported for the first time in the 1980s from the Scandinavian countries. Since then, it has been repeatedly encountered on needles from various European forest habitats. The abundance of these biofilms has been related to the climatic conditions, such as temperature and precipitation, as well as to the air pollution by nitrogen and sulfur oxides. Urban areas typically have relatively homogenous climates and profound variation in levels of air pollution. Therefore, variation in the occurrence of biofilms in localities within an urban area may be related to local anthropogenic air pollution. We investigated the abundance of biofilms occurring on needles of the common yew (Taxus baccata) in the city of Prague, Czech Republic. The biofilms were composed of algae, fungi and particulate matter. The cover area of the biofilms was marginally explained by a positive influence of short-term maximum atmospheric levels of nitrogen dioxide (NO2). The amounts of the microalgae were also positively influenced by short-term maximum NO2 levels. In addition, high atmospheric levels of particulate matter (PM10) were related to low abundance of algae. The microbial biofilms growing on widely cultivated conifers, such as the common yew, form one of the few commonly occurring natural communities in highly urbanized central areas of temperate European cities. Consequently, we propose that microscopic analysis of biofilms may be used as a rapid and cheap method to collect ecological data. Such data may be used in biomonitoring schemes illustrating the effects of anthropogenic air pollution on natural microcommunities in urban areas.

Ocean acidification and rising temperatures may increase biofilm primary productivity but decrease grazer consumption

Climate change may cause ecosystems to become trophically restructured as a result of primary producers and consumers responding differently to increasing CO2 and temperature. This study used an integrative approach using a controlled microcosm experiment to investigate the combined effects of CO2 and temperature on key components of the intertidal system in the UK, biofilms and their consumers (Littorina littorea). In addition, to identify whether pre-exposure to experimental conditions can alter experimental outcomes we explicitly tested for differential effects on L. littorea pre-exposed to experimental conditions for two weeks and five months. In contrast to predictions based on metabolic theory, the combination of elevated temperature and CO2 over a five-week period caused a decrease in the amount of primary productivity consumed by grazers, while the abundance of biofilms increased. However, long-term pre-exposure to experimental conditions (five months) altered this effect, with grazing rates in these animals being greater than in animals exposed only for two weeks. We suggest that the structure of future ecosystems may not be predictable using short-term laboratory experiments alone owing to potentially confounding effects of exposure time and effects of being held in an artificial environment over prolonged time periods. A combination of laboratory (physiology responses) and large, long-term experiments (ecosystem responses) may therefore be necessary to adequately predict the complex and interactive effects of climate change as organisms may acclimate to conditions over the longer term.

Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains

The social soil bacterium, Myxococcus xanthus, displays a variety of complex and highly coordinated behaviours, including social motility, predatory rippling and fruiting body formation. Here we show that M. xanthus cells produce a network of outer membrane extensions in the form of outer membrane vesicle chains and membrane tubes that interconnect cells. We observed peritrichous display of vesicles and vesicle chains, and increased abundance in biofilms compared with planktonic cultures. By applying a range of imaging techniques, including three-dimensional (3D) focused ion beam scanning electron microscopy, we determined these structures to range between 30 and 60 nm in width and up to 5 μm in length. Purified vesicle chains consist of typical M. xanthus lipids, fucose, mannose, N-acetylglucosamine and N-acetylgalactoseamine carbohydrates and a small set of cargo protein. The protein content includes CglB and Tgl outer membrane proteins known to be transferable between cells in a contact-dependent manner. Most significantly, the 3D organization of cells within biofilms indicates that cells are connected via an extensive network of membrane extensions that may connect cells at the level of the periplasmic space. Such a network would allow the transfer of membrane proteins and other molecules between cells, and therefore could provide a mechanism for the coordination of social activities.

Influence of mangrove zonation on CO2 fluxes at the sediment–air interface (New Caledonia)

Mangroves are the major ecosystems of tropical and subtropical coastlines. They are considered as a sink for atmospheric CO2 because they are characterized both by high net primary production, and by low rates of organic matter decomposition. However, a recent reassessment of the global mangrove budget suggests that organic carbon sinks have been underestimated, notably CO2 efflux from sediments and creek waters, and tidal export of dissolved inorganic carbon. Our objective was to understand the influence of mangrove zonation on the magnitude of CO2 fluxes at the sediment–air interface. Transparent and opaque dynamic closed chamber systems, coupled with an infra-red gas analyzer were used to measure CO2 fluxes. In addition, the physico-chemical properties (salinity, redox potential) of pore waters were determined, as well as the carbon content and the origin of surface sediments (Chlorophyll-a and δ13C). Depending on the type of measurement (in the dark with or without biofilm, in the light with biofilm) and mangrove stand (saltflat, Avicennia sp., or Rhizophora spp.), mean surface sediment CO2 fluxes ranged between 40±56 and 199±95mmol·m−2·d−1. We suggest that these differences mainly result both from the organic content and the redox conditions of the sediments, which are influenced by the physiological activities of the root system, and by the position and the elevation of the stand in the intertidal zone. In addition, the quality and abundance of biofilm, which also vary with the mangrove stand, also appear to strongly affect sediment CO2 fluxes as a result of chemical (metabolism) and also physical (barrier) processes.

Abundance of biofilm on intertidal rocky shores: Can trampling by humans be a negative influence?

Trampling by human visitors to rocky shores is a known stressor on macroorganisms. However, the effects of trampling on rocky intertidal biofilm, a complex association of microorganisms of ecological importance in coastal communities, have not been quantified. We evaluated the impact of trampling frequency and intensity on total biomass of epilithic microalgae on intertidal rocky shores in the southeast of Brazil. There was a trend of increase in the variability of biomass of biofilm in function of intensity of trampling, but no significant effects emerged among trampling treatments. The low influence of trampling on biofilm might be a result of the small dimensions of the organisms coupled with their natural resilience and roughness of the substrate; the former preventing the removal of biofilm layers by shoes and facilitating their quick recovery. Our results provide insights for management and conservation of coastal ecosystems revealing a weaker impact of trampling on biofilm than that reported on macroorganisms.

Grazing and nutrient effects on a high rocky intertidal of the Peruvian central coast

Bottom-up and top-down forces simultaneously influence benthic communities. However, their relative strength may vary with environmental conditions. In the Peruvian coast, the periwinkles are conspicuous grazers able to control both biofilm abundance and barnacle recruitment. Moreover, even in upwelling areas, algae living in the high intertidal may be nutrient-limited. Here we tested, in a nutrient-rich system, the relative effects of grazing and nutrient on the early succession of the high rocky intertidal. For this, we experimentally manipulated grazing and nutrient combined in a completely crossed design. Since nutrient and grazing seem to alter benthic community at different temporal scales, their effects were tested over the long-term (14months) and the short-term (1months). Over the long-term, barnacle cover increased throughout time, but was neither affected by grazing nor fertilization. At all sampling times fertilization increased biofilm cover and reduced bare rock, while exclusion of grazers also enhanced biofilm. The effect of grazing on bare rock varied through the succession. Macroalgae reached a maximum cover of 6.88%. Over the short-term, chlorophyll-a content was not affected by nutrient addition but was always higher in plots where grazer were excluded. Our results suggest that biofilm might be nutrient-limited in the high intertidal, despite being located in an upwelling area. However, the effects of grazers and nutrients on the biofilm are not reflected in macroalgae or barnacles, probably because of the occurrence of other structuring factors. In the short-term, grazing seems to have greater effect on biofilm, irrespective of the nutrient level, whereas over the longer-term, biofilm can take advantage of nutrient enhancement only in the absence of grazers.

Variability in Foraging Behavior and Implications for Diet Breadth among Semipalmated Sandpipers Staging in the Upper Bay of Fundy

During its fall migration stopover on mudflats in the upper Bay of Fundy, Canada, the Semipalmated Sandpiper (Calidris pusilla) is thought to feed primarily on the amphipod Corophium volutator (mudshrimp). Semipalmated Sandpipers typically use a peck-probe foraging strategy and, until recently, there had been little evidence of variability or opportunism in their foraging habits during this stopover. From 2006 to 2008 we recorded data on the sandpipers' foraging behavior and food availability at three commonly used mudflats. Behavior and food availability varied considerably at one site in one year. In 2006 at Grande Anse, where mudshrimp densities were exceptionally low and ostracod densities very high, the peck-probe strategy was almost completely abandoned for “skimming,” a foraging behavior novel in this species. Because of similarities between skimming and grazing, used by Western Sandpipers (Calidris mauri) to consume biofilm, we hypothesized that birds switched to skimming to feed on biofilm. However, chlorophyll a concentration in the top 2–3 mm of sediment, an index of biofilm abundance, was not a good predictor of proportion of time spent skimming. Instead, skimming had a strong, positive relationship with ostracod density, suggesting that the sandpipers skimmed opportunistically to feed on ostracods rather than to target biofilm. Thus Semipalmated Sandpipers are capable of adapting to changes at traditional staging areas by using novel foraging mechanisms, apparently to forage opportunistically on alternative prey. If staging habitats continue to change, alternative foods and foraging modes may become increasingly important to the success of this species' migration.

Ecosystem response to a salmon disturbance regime: Implications for downstream nutrient fluxes in aquatic systems

Recent work in salmon spawning streams has shown that sediment resuspended during nest construction flocculates with salmon organic matter to form suspended composite particles characterized by increased size and settling velocities. In a river system, these flocs have the potential to interact with benthic biofilms, suggesting a mechanism for the incorporation of organic matter into aquatic food webs. Using the Horsefly River spawning channel in central British Columbia, the spatial scale of biofilm floc trapping was evaluated for a salmon disturbance regime, which consists of the active digging of redds, spawning, and carcass decay. We stocked two sequential enclosures in the spawning channel with sockeye salmon (Oncorhynchus nerka) and established one upstream control enclosure. Biofilms were sampled for chlorophyll a, trapped sediment, and marine isotope tracers (δ15N and δ13C). In the active‐spawn period, biofilm abundance was reduced due to spawning disturbance, with isotope values indicating low utilization of marine‐derived nutrients (MDNs). During the post‐spawn period, downstream biofilm abundance exceeded pre‐spawn values, indicating a near‐field nutrient pulse with isotope values reflecting biofilm utilization of MDNs. At the same time, an increase in biofilm trapping efficiency occurred in concert with a significant increase in the in situ particle size of suspended sediment, suggesting that flocs were a temporary storage site of MDNs. The retention of MDNs over short spatial scales acts to retard the flushing of MDNs to downstream rearing lakes. The magnitude of these processes has ecological implications on the downstream lake's productivity, thereby influencing the success of future salmon stocks.

Gene expression and deletion analysis of mechanisms for electron transfer from electrodes to Geobacter sulfurreducens

Geobacter sulfurreducens is one of the few microorganisms available in pure culture known to directly accept electrons from a negatively poised electrode. Microarray analysis was used to compare gene transcript abundance in biofilms of G. sulfurreducens using a graphite electrode as the sole electron donor for fumarate reduction compared with transcript abundance in biofilms growing on the same material, but not consuming current. Surprisingly, genes for putative cell-electrode connections, such as outer-surface cytochromes and pili, which are highly expressed in current-producing biofilms, were not highly expressed in current-consuming biofilms. Microarray analysis of G. sulfurreducens gene transcript abundance in current-consuming biofilms versus current-producing biofilms gave similar results. In both comparative studies current-consuming biofilms had greater transcript abundance for a gene (GSU3274) encoding a putative monoheme, c-type cytochrome. Deletion of genes for outer-surface proteins previously shown to be essential for optimal electron transfer to electrodes had no impact on electron transfer from electrodes. Deletion of GSU3274 completely inhibited electron transfer from electrodes, but had no impact on electron transfer to electrodes. These differences in gene expression patterns and the impact of gene deletions suggest that the mechanisms for electron transfer from electrodes to G. sulfurreducens differ significantly from the mechanisms for electron transfer to electrodes.

Biofilm bacterial communities and abundance in a full-scale drinking water distribution system in Shanghai

Community diversity and abundance of biofilms from a full-scale drinking water distribution system in Shanghai were characterized by denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA sequences and heterotrophic plate count (HPC), respectively. Bacteria affiliated to the Beta- and Gamma-Proteobacteria were dominating in both in-situ and HPC-culturable bacterial communities. Other bacteria present included members of Alphaproteobacteria, Bacteroides, Actinobacteria, Nitrospirae and Firmicutes. Acidovorax, Ralstonia and Acinetobacter were common species in biofilms. Klebsiella pneumoniae and Enterobacter sp. were detected in the local distribution system. Dissolved organic carbon (DOC), residual disinfectant and temperature were the most important factors influencing both bacterial abundance and composition. HPC for biofilm sample was not correlated with its community diversity.

Colonization dynamics of biofilm-associated ciliate morphotypes at different flow velocities

The impact of flow velocity on initial ciliate colonization dynamics on surfaces were studied in the third order Ilm stream (Thuringia, Germany) at a slow flowing site (0.09 m s −1) and two faster flowing sites (0.31 m s −1) and in flow channels at 0.05, 0.4, and 0.8 m s −1. At the slow flowing stream site, surfaces were rapidly colonized by ciliates with up to 60 cells cm −2 after 24 h. In flow channels, the majority of suspended ciliates and inorganic matter accumulated at the surface within 4.5 h at 0.05 m s −1. At 0.4 m s −1 the increase in ciliate abundance in the biofilm was highest between 72 and 168 h at about 3 cells cm −2 h −1. Faster flow velocities were tolerated by vagile flattened ciliates that live in close contact to the surface. Vagile flattened and round filter feeders preferred biofilms at slow flow velocities. Addition of inorganic particles (0, 0.6, and 7.3 mg cm −2) did not affect ciliate abundance in flow channel biofilms, but small ciliate species dominated and number of species was lowest (16 species cm −2) in biofilms at high sediment content. Although different morphotypes dominated the communities at contrasting flow velocities, all functional groups contributed to initial biofilm communities implementing all trophic links within the microbial loop.

TIMBER HARVEST TRANSFORMS ECOLOGICAL ROLES OF SALMON IN SOUTHEAST ALASKA RAIN FOREST STREAMS

Although species commonly modify habitats and thereby influence ecosystem structure and function, the factors governing the ecological importance of these modifications are not well understood. Pacific salmon have repeatedly been shown to positively influence the abundance of benthic biota by annually transferring large quantities of nutrients from marine systems to the nutrient-poor freshwaters in which they spawn. Conversely, other studies have demonstrated that salmon can negatively influence the abundance of freshwater biota, an effect attributed to bioturbation during upstream migration and nest construction. The factors determining which of these contrasting ecological effects predominates are unknown, including how human activities, such as land use, influence ecological responses to salmon. We sampled a key basal food resource, sediment biofilm, in seven southeast Alaskan streams impacted to varying degrees by timber harvest. Biofilm abundance (measured as chlorophyll a and ash-free dry mass) was positively related to timber-harvest intensity prior to salmon arrival. However, during the salmon run, an inverse relationship emerged of more abundant biofilm in less-harvested watersheds. Among-stream variability in biofilm response to salmon was largely explained by sediment particle size, which was larger in less-harvested watersheds. Collectively, these results suggest that, by altering stream sediment size, timber harvest transformed the dominant effect of salmon from nutrient enrichment to physical disturbance, thus modifying nutrient linkages between marine and freshwater ecosystems.

Grazing effects of the periwinkle Echinolittorina peruviana at a central Peruvian high rocky intertidal

Echinolittorina peruviana is the most common gastropod in the high intertidal zone of Peru, representing more than 80% of the individuals present at that zone. Experimental removal of snails was used to evaluate their effects on (a) abundance of epilithic biofilm, (b) barnacle recruitment, and (c) abundance of macroalgae under “normal” conditions of the El Nino Southern Oscillation (ENSO). Experiments were carried out from October 2005 to April 2007 at two intertidal levels of a semi-protected rocky shore of central Peru. Results demonstrated that E. peruviana is able to control biofilm abundance and barnacle recruitment at both heights investigated, with marked effects in the lower zone. Erect macroalgae (Ulva spp. and Gelidium spp.) were less affected by grazing; but negative effects were observed on macroalgal crusts. Season and physical stress seem to play a more important role in the abundance of macroalgae in the high intertidal. Our results are similar to those reported elsewhere for high shore littorinids and represent baseline data to understand how the role of intertidal consumers will vary under the cold (La Nina) and warm (El Nino) phases of ENSO on these shores.

Attachment of Balanus amphitrite larvae to biofilms originating from contrasting environments

This study examined the attachment response of Balanus amphitrite larvae to bacteria- dominated biofilms originating from 4 sites of varying environmental conditions in the intertidal region of subtropical Hong Kong waters and during 2 seasons (winter and summer), under both laboratory and field conditions. Using multiple fingerprinting techniques (terminal restriction frag- ment length polymorphism, denaturing gradient gel electrophoresis and fluorescence in situ hybri- dization), we observed differences in the bacterial community composition of biofilms originating from the 4 sites. These biofilm samples were used to study the linkage between spatial changes in bacterial communities of biofilms and larval choice at the time of attachment. It was hypothesized that cyprids can distinguish biofilms originating from habitats that support higher recruitment. Both laboratory and field multiple-choice bioassays demonstrated that cyprids preferred to attach on biofilms originating from habitats where recruitment, juvenile growth and survival were the highest, thereby accepting the hypothesis proposed. This study did not identify particular bacterial species or groups in biofilms that attract or inhibit larval attachment, but we could correlate site-specific varia- tions in bacterial community composition with larval choice, whereas bacterial abundance in biofilms was less important in this regard. Overall, this study highlights the significance of site-specific variation in biofilms on larval recruitment and demonstrated the discriminative behavior of barnacle larvae to biofilms originating from contrasting environments in the intertidal region. Thus, attach- ment cues from biofilms may also play a significant role in generating spatial variation in larval recruitment.

Recolonization by the benthos of an acid stream following a drought

This study investigates recolonization by benthic algae and macroinvertebrates following a drought and subsequent drying of an acid stream in southern England, normally characterised by permanent flows. Re-establishment of epilithic biofilm on the upper surfaces of mineral substrata was tracked in a shallow riffle between Sep. 1995 and Mar. 1996. Recolonization of these surfaces by macroinvertebrates was also followed, as was their benthic density. Post-drought abundance of biofilm and invertebrates was compared with that of the pre-drought year (Aug 1994-Mar 1995). We found that viable algal cells remained attached to the dry substratum despite 9 weeks of drying. On rewetting, these resistant benthic algal populations increased in biomass (evidenced by AFDM, chlorophyll density) and cell density, until a series of high flow events scoured the stones in winter 1995/1996. Spatial variation in the biofilm also existed: generally, algal biomass was higher on bedrock than on small or large stones. Recolonization of the previously dry stream bed by macroinvertebrates was rapid; after 3 days of flow, 665 larvae/m 2 , composed of a mean of 7.5 taxa, had been attained. The total numbers of invertebrates peaked 38 days after rewetting when a density of 2185 larvae/m 2 was found. This increase in abundance was mainly driven by the tanypod chironomid Zavrelimyia sp., which represented 56 % of total numbers on day 38. Detrended correspondence analysis (DCA) revealed that, as colonization time progressed, the macroinvertebrate community became progressively more dissimilar to the pre-drought community, until high flows occurred in winter 1996 and community composition finally converged to that characteristic of the pre-drought community. Recovery following drought was rapid despite the depauperate and unproductive nature of this acid stream.

A technique To quantify the population size and composition of the biofilm component in communities of bacteria in the phyllosphere

The presence of microbial biofilms in the phyllosphere of terrestrial plants has recently been demonstrated, but few techniques to study biofilms associated with living plant tissues are available. Here we report a technique to estimate the proportion of the bacterial population on leaves that is assembled in biofilms and to quantitatively isolate bacteria from the biofilm and nonbiofilm (solitary) components of phyllosphere microbial communities. This technique is based on removal of bacteria from leaves by gentle washing, separation of biofilm and solitary bacteria by filtration, and disintegration of biofilms by ultrasonication. The filters used for this technique were evaluated for their nonspecific retention rates of solitary bacteria and for the efficiency of filtration for different concentrations of solitary bacteria in the presence of biofilms and other particles. The lethality and efficiency of disintegration of the sonication conditions used here were also evaluated. Isolation and quantification of bacteria by this technique is based on use of culture media. However, oligonucleotide probes, sera, or epifluorescent stains could also be used for direct characterization of the biofilm and solitary bacteria in the suspensions generated by this technique. Preliminary results from estimates of biofilm abundance in phyllosphere communities show that bacteria in biofilms constitute between about 10 and 40% of the total bacterial population on broad-leaf endive and parsley leaves.