Desmids and Biofilms of Freshwater Wetlands: Development and Microarchitecture

Freshwater wetlands constitute important ecosystems, and their benthic, attached microbial communities, including biofilms, represent key habitats that contribute to primary productivity, nutrient cycling, and substrate stabilization. In many wetland biofilms, algae constitute significant parts of the microbial population, yet little is known about their activities in these communities. An analysis of wetland biofilms from the Adirondack region of New York (USA) was performed with special emphasis on desmids, a group of evolutionarily advanced green algae commonly found in these habitats. Desmids constituted as much as 23.7% of the total algal and cyanobacterial flora of the biofilms during the July and August study periods. These algae represented some of the first eukaryotes to colonize new substrates, and during July their numbers correlated with fluctuations in general biofilm parameters such as biofilm thickness and dry weight as well as total carbohydrate. Significant numbers of bacteria were associated with both the EPS sheaths and cell wall surfaces of the desmids. Colonization of new substrates and development of biofilms were rapid and were followed by various fluctuations in microbial community structure over the short- and long-term observations. In addition to desmids, diatoms, filamentous green algae and transient non-motile phases of flagellates represented the photosynthetic eukaryotes of these biofilms.     

Diatoms in Acid Mine Drainage and Their Role in the Formation of Iron-Rich Stromatolites

Adverse conditions in the acid mine drainage (AMD) system at the Green Valley mine, Indiana, limit diatom diversity to one species, Nitzschia tubicola. It is present in three distinct microbial consortia: Euglena mutabilis-dominated biofilm, diatomdominated biofilm, and diatom-exclusive biofilm. E. mutabilis dominates the most extensive biofilm, with lesser numbers of N. tubicola, other eukaryotes, and bacteria. Diatom-dominated biofilm occurs as isolated patches containing N. tubicola with minor fungal hyphae, filamentous algae, E. mutabilis, and bacteria. Diatom-exclusive biofilm is rare, composed entirely of N. tubicola.

Diatom distribution is influenced by seasonal and intraseasonal changes in water temperature and chemistry. Diatoms are absent in winter due to cool water temperatures. In summer, isolated patchy communities are present due to warmer water temperatures. In 2001, the diatom community expanded its distribution following a major rainfall that temporarily diluted the effluent, creating hospitable conditions for diatom growth. After several weeks when effluent returned to preexisting conditions, the diatom biofilm retreated to isolated patches, and E. mutabilis biofilm flourished.

Iron-rich stromatolites underlie the biofilms and consist of distinct laminae, recording spatial and temporal oscillations in physicochemical conditions and microbial activity. The stromatolites are composed of thin, wavy laminae with partially decayed E. mutabilis biofilm, representing microbial activity and iron precipitation under normal AMD conditions. Alternating with the wavy layers are thicker, porous, spongelike laminae composed of iron precipitated on and incorporated into radiating colonies of diatoms. These layers indicate episodic changes in water chemistry, allowing diatoms to temporarily dominate the system.     

Comparative effect of temperature on biofilm formation in natural and modified marine environment

Progression of biofilm formation was monitored at two stations near a nuclear power plant, Kalpakkam, located near coastal waters of Bay of Bengal. These stations are natural marine environment, station 1; and the condenser outfall area of the power plant the modified marine environment station 2. The biofilm formed on plexiglas panels was analysed in triplicates at 24 h intervals for various physical, chemical and biological parameters for 120 h (5 days). The biofilm formation showed both temporal and spatial variation in various parameters assayed. Among the water-quality parameters analysed, seawater temperature showed significant increase (~5°C) at station 2. The increase in water temperature enhanced the metabolism and influenced most of the biofilm parameters assayed at station 2. Biofilm formed at station 2 was very thick (113 μm) than that of at station 1 (22 μm). The distribution of parameters like biofilm thickness, biomass, chlorophyll a, particulate organic carbon, hexose sugar and diatom counts showed similar trend (i.e., a sharp increase after 96 h of biofilm growth) in the biofilm formed at station 2. Moderately high ammonia levels (44 μg l⁻¹) were detected in the biofilm formed at station 2. The biofilm microbiota was diverse at both the stations: it constituted bacteria [nitrate reducers (NRB), ammonia oxidizers (AOB) and culturable aerobic heterotrophic bacteria (CAHB)], algae and macrofoulants. The various bacterial types assayed showed a population range from 10² to 10⁶ cfu cm⁻². The final community after 120 h at station 1 comprised CAHB, NRB, diatoms, barnacle cyprids and juvenile bryozoans. At station 2, the biofilm initially consisted of CAHB, NRB and diatoms but after 120 h, AOB, cyanobacteria and filamentous algae were dominant. The plausible factors that influenced biofilm formation were temperature, nutrients and organic matter. The biofilm phenomenon in natural and modified marine environment was hypothesized and discussed.     

Phylogenetic Composition, Spatial Structure, and Dynamics of Lotic Bacterial Biofilms Investigated by Fluorescent in Situ Hybridization and Confocal Laser Scanning Microscopy

Abstract The phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Biofilms grew on independently removable polycarbonate slides exposed in the reactor system with natural river water as inoculum and sole nutrient and carbon source. The microbial biofilm community developed from attached single cells and distinct microcolonies via a more confluent structure characterized by various filamentous bacteria to a mature biofilm rich in polymeric material with fewer cells on a per-area basis after 56 days. During the different stages of biofilm development, characteristic microcolonies and cell morphotypes could be identified as typical features of the investigated lotic biofilms. In situ analysis using a comprehensive suite of rRNA-targeted probes visualized individual cells within the alpha-, beta-, and gamma-Proteobacteria as well as the Cytophaga–Flavobacterium group as major parts of the attached community. The relative abundance of these major groups was determined by using digital image analysis to measure specific cell numbers as well as specific cell area after in situ probing. Within the lotic biofilm community, 87% of the whole bacterial cell area and 79% of the total cell counts hybridized with a Bacteria specific probe. During initial biofilm development, beta-Proteobacteria dominated the bacterial population. This was followed by a rapid increase of alpha-Proteobacteria and bacteria affiliated to the Cytophaga–Flavobacterium group. In mature biofilms, alpha-Proteobacteria and Cytophaga–Flavobacteria continued to be the prevalent bacterial groups. Beta-Proteobacteria constituted the morphologically most diverse group within the biofilm communities, and more narrow phylogenetic staining revealed the importance of distinct phylotypes within the beta1-Proteobacteria for the composition of the microbial community. The presence of sulfate-reducing bacteria affiliated to the Desulfovibrionaceae and Desulfobacteriaceae confirmed the range of metabolic potential within the lotic biofilms. Received: 24 September 1998; Accepted: 17 February 1999     

Quantification of diatoms in biofilms: Standardisation of methods

Abstract

Benthic diatoms, which often dominate marine biofilms are mostly pennate along with a few centric species that have an attached mode of life. Even though the range of diatoms in biofilms is diverse, their ecology is poorly understood because of the difficulty in sampling and enumeration. Scraping or brushing are the traditional methods used for removal of diatoms from biofilms developed on solid substrata. The method of removal is the most critical step in enumerating the biofilm diatom community structure. In this study, a nylon brush and ceramic scraper were used as tools for the removal of diatoms from 1 – 4-day-old biofilms developed on fibreglass coupons and glass microscope slides. Standardisation of methods showed that the sample volume used in the analyses had the least influence on the quantification, whereas the method of removal was critical. The nylon brush was more efficient at recovering diatoms compared to a ceramic scraper. Direct microscopic enumeration of the community in the case of glass slides indicated that scraping resulted in between 30–50% underestimation. Heterogeneity in diatom community structure between replicate samples is one possible reason for such underestimation.     

Microbial Dynamics of an Epilithic Mat Community in a High Alpine Stream

Studies were conducted to examine interrelationships between the heterotrophic and phototrophic populations within an epilithic community in the outlet stream of a high alpine lake. Levels of nitrates, phosphates, and total organic compounds in the lake were consistently near the lower limits of detectability. Microscopic examination of the community by phase-contrast light microscopy and scanning electron microscopy revealed diatoms, filamentous algae, and bacteria embedded within a dense gelatinous matrix. Chlorophyll a and primary productivity measurements had peak values in early August, with subsequent declines. Bacterial heterotrophic activity, as measured by V_max, turnover rate, and relative activity, increased significantly as the phototrophic community declined. This trend in heterotrophic activity was not accompanied by an increase in total bacterial numbers as determined by epi-illuminated fluorescence microscopy. These results suggest that the phototrophic community responded to changes in, or interactions among, various chemical and physical factors throughout the study period. The catabolic activity of the sessile bacteria appeared to be positively influenced by changes in the mat environment resulting from the decline of the phototrophic populations.     

Structural and Functional Dynamics of Sulfate-Reducing Populations in Bacterial Biofilms

We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 μm during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10⁸ SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that Desulfobulbus and Desulfovibrio were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different Desulfobulbus and Desulfovibrio species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.     

Succession and physiological health of freshwater microalgal fouling in a Tasmanian hydropower canal

Freshwater microalgal biofouling in hydropower canals in Tarraleah, Tasmania, is dominated by a single diatom species, Gomphonema tarraleahae. The microfouling community is under investigation with the aim of reducing its impact on electricity generation. Species succession was investigated using removable glass slides. Fouled slides were examined microscopically and for chlorophyll a biomass. Chl a biomass increased steeply after 8 weeks (0.09–0.87 mg m^?2), but increased much earlier on slides surrounded by a biofouled inoculum. Succession began with low profile diatoms such as Tabellaria flocculosa, progressing to stalked diatoms such as Gomphonema spp. and Cymbella aspera. Few chlorophytes and no filamentous algae were present. Pulse amplitude modulated fluorometry was used to measure the physiological health of fouling on the canal wall. Maximum quantum yield (F _v/F _m) measurements were consistently <0.18, indicating that the fouling mat consisted of dead or dying algae. The succession and physiological health of cells in the fouling community has broad implications for mitigation techniques used.     

Effects of initial surface wettability on biofilm formation and subsequent settlement of Hydroides elegans

Hydroides elegans is a major fouling organism in tropical waters around the world, including Pearl Harbor, Hawaii. To determine the importance of initial surface characteristics on biofilm community composition and subsequent colonization by larvae of H. elegans, the settlement and recruitment of larvae to biofilmed surfaces with six different initial surface wettabilities were tested in Pearl Harbor. Biofilm community composition, as determined by a combined approach of denaturing gradient gel electrophoresis and fluorescence in situ hybridization, was similar across all surfaces, regardless of initial wettability, and all surfaces had distinct temporal shifts in community structure over a 10 day period. Larvae settled and recruited in higher numbers to surfaces with medium to low wettability in both May and August, and also to slides with high wettability in August. Pearl Harbor biofilm communities developed similarly on a range of surface wettabilities, and after 10 days in Pearl Harbor all surfaces were equally attractive to larvae of Hydroides elegans, regardless of initial surface properties.     

Biofilm diatom community structure: influence of temporal and substratum variability

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.     

Dynamics of grazing protozoa follow that of microalgae in natural biofilm communities

This study investigates the dynamics of protozoan community in biofilms formed on inert artificial surfaces suspended in various freshwater environments. The results also test the hypothesis that the dynamics of protozoan and microalgal communities in biofilms are interdependent because the latter form one of the major food items of benthic protozoa. Cleaned glass slides were suspended in surface waters at four sampling locations to collect biofilm communities. The glass slides after retrieval were observed under a microscope for diatom and protozoan density and their generic composition. Members of protozoa belonging to phylum Sarcomastigophora dominated the protozoan community followed by phylum Ciliophora in all sampling locations. The variation of protozoan feeding groups showed an initial abundance of autotrophs/holophytes which gave way to heterotrophs, predators, and bacterivores towards the end of the study. The density and generic composition of protozoa varied significantly with the age of biofilm and sampling location. The density variation of protozoa followed that of diatoms in all four sampling locations and this has resulted in a significant positive correlation between diatom and protozoan densities. This suggests the dependency and/or food web connectedness of these two communities in natural biofilms.     

Effects of seawater ozonation on biofilm development in aquaculture tanks

Microbial biofilms developing in aquaculture tanks represent a reservoir for opportunistic bacterial pathogens, and procedures to control formation and bacterial composition of biofilms are important for the development of commercially viable aquaculture industries. This study investigated the effects of seawater ozonation on biofilm development on microscope glass slides placed in small-scale aquaculture tanks containing the live feed organism Artemia. Fluorescence in situ hybridization (FISH) demonstrated that ozonation accelerated the biofilm formation cycle, while it delayed the establishment of filamentous bacteria. Gammaproteobacteria and Alphaproteobacteria were the most abundant bacterial groups in the biofilm for both water types, but ozonation influenced their dynamics. With ozonation, the bacterial community structure was relatively stable and dominated by Gammaproteobacteria throughout the experiment (21–66% of total bacteria). Without ozonation, the community showed larger fluctuations, and Alphaproteobacteria emerged as dominant after 18 days (up to 54% of total bacteria). Ozonation of seawater also affected the dynamics of less abundant populations in the biofilm such as Betaproteobacteria, Planctomycetales and the Cytophaga/Flavobacterium branch of phylum Bacteroidetes. The abundance of Thiothrix, a bacterial genus capable of filamentous growth and fouling of larvae, increased with time for both water types, while no temporal trend could be detected for the genus Vibrio. Denaturing gradient gel electrophoresis (DGGE) demonstrated temporal changes in the dominant bacterial populations for both water types. Sequencing of DGGE bands confirmed the FISH data, and sequences were related to bacterial groups commonly found in biofilms of aquaculture systems. Several populations were closely related to organisms involved in sulfur cycling. Improved Artemia survival rates in tanks receiving ozonated water suggested a positive effect of ozonation on animal health. Although the used ozonation protocol did not hinder biofilm formation, the results suggest ozonation as a promising approach for manipulation of bacterial populations in aquaculture systems, which can prove beneficial for cultured animals.     

Quantification of diatoms in biofilms: standardisation of methods

Benthic diatoms, which often dominate marine biofilms are mostly pennate along with a few centric species that have an attached mode of life. Even though the range of diatoms in biofilms is diverse, their ecology is poorly understood because of the difficulty in sampling and enumeration. Scraping or brushing are the traditional methods used for removal of diatoms from biofilms developed on solid substrata. The method of removal is the most critical step in enumerating the biofilm diatom community structure. In this study, a nylon brush and ceramic scraper were used as tools for the removal of diatoms from 1 - 4-day-old biofilms developed on fibreglass coupons and glass microscope slides. Standardisation of methods showed that the sample volume used in the analyses had the least influence on the quantification, whereas the method of removal was critical. The nylon brush was more efficient at recovering diatoms compared to a ceramic scraper. Direct microscopic enumeration of the community in the case of glass slides indicated that scraping resulted in between 30-50% underestimation. Heterogeneity in diatom community structure between replicate samples is one possible reason for such underestimation.     

Optimal colonization and growth of marine benthic diatoms on artificial substrata: protocol for a routine use in bioindication

Benthic diatoms growing on hard substrata are used for their bioindication ability in freshwater quality monitoring. Artificial substrata are needed in cases where any natural substrate is present or to achieve similar sampling conditions between sites. Prior to use marine benthic diatoms for monitoring, a standardized protocol for sampling on artificial substrata must be set up. Two major types of information are required: (1) the time needed for a diatom community to be well developed and mature (climax stage); (2) the optimal growth conditions, given that the substrataum nature and texture are important parameters for the initial phase of biofilm development and can influence the future diatom assemblage. Three substrataum types were tested: frosted Plexiglass®, frosted glass, and rough enameled tiles. They were submerged for 8 weeks and sampled weekly. The experiment was conducted at five sites of distinct morphology and water chemistry, along the coastal area of Martinique Island, French West Indies. Development of diatom community was studied through biofilm dry weight, valve density, species richness, and species relative abundances. Globally, substratum type had no significant effect on any parameter. Frosted Plexiglass® was found to be the most interesting substratum because of higher valve densities and practical use. The asymptotic phase of biofilm development was encountered between 5 and 8 weeks depending on site and parameter. A compromise between community development and vandalism or loss through time was fixed to 5 weeks. This period is longer than for stream environments and is valid for tropical oligotrophic marine environments.     

Recruitment of Antarctic marine eukaryotes onto artificial surfaces

Activities related to Antarctic research stations have caused significant local impacts on the marine environment, potentially affecting the recruitment of benthic invertebrates. Herein, we report the community structure of recruiting marine eukaryotes onto artificial substrata using molecular techniques. Slides were deployed at three sites adjacent to McMurdo Station, Scott Base, and Cape Armitage in McMurdo Sound. Denaturing gradient gel electrophoresis (DGGE) analysis revealed complex and diverse eukaryotic communities had established on artificial surfaces deployed at a range of site and depth regimes after 12 months. Analysis of similarity results detected significantly greater variability in community profiles among sites than within sites. The nonmetric multidimensional scaling plot constructed from DGGE banding patterns revealed different benthic communities had established at 12 and 18 m depths. Despite this, the variation in community composition was greater among sites than between depths, especially at Cape Armitage and Scott Base. Sequence analysis of excised DGGE bands revealed a predominance of arthropod and dinoflagellate sequences at Cape Armitage. In contrast, a wide diversity of phyla including cnidaria, bryozoa, protozoa, dinoflagellates, arthropods, platyhelminths, and annelids were present adjacent to the two research stations. The abundance of diatoms detected in Cape Armitage benthic assemblages exceeded the abundance of diatoms from McMurdo Station and Scott Base by almost two orders of magnitude. The discovery that distinct eukaryotic communities recruit at different sites and depths is probably due to complex interactions between multiple factors including water quality, larval supply, and light. The detection of sessile phyla on slides at each of the sites indicates that the pollution profiles present at each site is not an impediment to successful recruitment of these species.     

Long‐Term Dynamics of Microbial Biofilm Communities of the River Rhine with Special References to Ciliates

The aim of this study was to quantify and qualify seasonal changes of all important components of a microbial biofilm community. We explored the development of the biofilm community structure on submerged glass slides for 15 months including all organisms from bacteria to macro‐invertebrates. Besides bacteria, heterotrophic flagellates were the most abundant biofilm component followed by ciliates, meiofauna organisms and algae. Most important were sessile choanoflagellates, peritrichous ciliates and some crustaceans. Ciliates and macrofauna were the most important components with regard to the total biovolume. The biofilm architecture was strongly influenced by extracellular structures produced by protozoans and macro‐invertebrates. Alterations within the biofilm community were mainly due to changes in abundances rather than in the composition except for heterotrophic flagellates and macro‐invertebrates. Biofilm organisms were dominated by planktivorous organisms exerting a strong grazing impact on the plankton organisms in this large river. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The development of marine biofilms on two commercial non-biocidal coatings: a comparison between silicone and fluoropolymer technologies

The antimicrobial performance of two fouling-release coating systems, Intersleek 700? (IS700; silicone technology), Intersleek 900? (IS900; fluoropolymer technology) and a tie coat (TC, control surface) was investigated in a short term (10 days) field experiment conducted at a depth of ca 0.5 m in the Marina Bandar Rawdha (Muscat, Oman). Microfouling on coated glass slides was analyzed using epifluorescence microscopy and adenosine-5'-triphosphate (ATP) luminometry. All the coatings developed biofilms composed of heterotrophic bacteria, cyanobacteria, seven species of diatoms (2 species of Navicula, Cylindrotheca sp., Nitzschia sp., Amphora sp., Diploneis sp., and Bacillaria sp.) and algal spores (Ulva sp.). IS900 had significantly thinner biofilms with fewer diatom species, no algal spores and the least number of bacteria in comparison with IS700 and the TC. The ATP readings did not correspond to the numbers of bacteria and diatoms in the biofilms. The density of diatoms was negatively correlated with the density of the bacteria in biofilms on the IS900 coating, and, conversely, diatom density was positively correlated in biofilms on the TC. The higher antifouling efficacy of IS900 over IS700 may lead to lower roughness and thus lower fuel consumption for those vessels that utilise the IS900 fouling-release coating.     

Biofilm diatom community structure: Influence of temporal and substratum variability

Abstract

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.     

Biostimulation of Estuarine Microbiota on Substrate Coated Agar Slides: A Novel Approach to Study Diversity of Autochthonous Bdellovibrio- and Like Organisms

Characterization of Bdellovibrio- and like organisms (BALOs) from environmental samples involves growing them in the presence of Gram-negative prey bacteria and isolation of BALO plaques. This labor-intensive enrichment and isolation procedure may impede the detection and phylogenetic characterization of uncultivable BALOs. In this article, we describe a simple slide biofilm assay to improve detection and characterization of BALO microbiota. Agar spiked with biostimulants such as yeast extract (YE), casamino acids (CA), or concentrated cells of Vibrio parahaemolyticus P5 (most widely used prey bacteria for isolation of halophilic BALOs) was plated onto buffed glass slides and exposed to water samples collected from Apalachicola Bay, Florida. After incubating for a week, diversity of the biofilm bacterial community was studied by culture-dependent and culture-independent molecular methods. The results revealed that most probable numbers (MPNs) of BALOs and total culturable bacteria recovered from YE agar slide were significantly higher than the numbers on CA- or P5-spiked agar slides. Polymerase chain reaction–restriction fragment length polymorphism followed by 16S rDNA sequencing of clones from different biostimulants resulted in identification of a plethora of Gram-negative bacteria predominantly from the alpha, gamma, delta-proteobacteria, and the Cytophaga–Flavobacterium–Bacteroides group. Corresponding to the higher biomass on the YE agar slide, the BALO clone library from YE was most diverse, consisting of Bacteriovorax spp. and a novel clade representing Peredibacter spp. Microbiota from all three biostimulated biofilms were exclusively Gram-negative, and each bacterial guild represented potential prey for BALOs. We propose the use of this simple yet novel slide biofilm assay to study oligotrophic aquatic bacterial diversity which could also potentially be utilized to isolate marine bacteria with novel traits.