Apparent Surface Associated Lag Time in Growth of Primary Biofilm Cells

Abstract The ability of microorganisms to form biofilms has been well documented. Bacterial cells make a transition from a planktonic state to a sessile state, replicate, and subsequently populate a surface. In this study, organisms that initially colonize a ``clean' surface are referred to as ``primary' biofilm cells. The progeny of the first generation of sessile cells are known as ``secondary' biofilm cells. This study examined the growth of planktonic, primary, and secondary biofilm cells of a green fluorescent protein producing (GFP+) Pseudomonas aeruginosa PA01. Biofilm experiments were performed in a parallel plate flow cell reactor with a glass substratum. Individual cells were tracked over time using a confocal scanning laser microscope (CSLM). Primary cells experience a lag in their growth that may be attributed to adapting to a sessile environment or undergoing a phenotypic change. This is referred to as a surface associated lag time. Planktonic and secondary biofilm cells both grew at a faster rate than the primary biofilm cells under the same nutrient conditions. Received: 17 September 1999; Accepted: 13 January 2000; Online Publication: 25 April 2000     

Functional and Structural Responses of a Degradative Microbial Community to Substrates with Varying Degrees of Complexity in Chemical Structure

Abstract The objective of the present study was to determine whether cultivation of a degradative community on substrates with varying degrees of chlorination and complexity in chemical structure, as well as cultivation in batch and flow cell culture, would alter the community's functional capability. The community was isolated from oil-contaminated soil and maintained in the laboratory on 2,4,6-trichlorobenzoic acid for 5 months before its ability to grow on 15 different chemicals as sole carbon source was evaluated in batch and flow cell systems. While the community could grow and develop biofilms in flow cells on all the substrates, only 11 of the 15 substrates could support growth in batch culture. Although biofilm development was less extensive on chemicals such as pentachlorophenol (2.09% average area covered by biofilm; average biofilm depth = 3 μm) than on 2,4,6-trichlorobenzoic acid (50.84% area covered; biofilm depth = 6.4 μm), no correlation was observed between the degree of chlorination, or number of rings, and the number of planktonic cells or biofilm biomass. In contrast, physicochemical characteristics such as the octanol/water partition coefficient had a significant effect on the development of biofilm biomass. In the case of planktonic communities, the degree of chlorination and ring number also had no effect on the BIOLOG carbon utilization profiles of the resulting communities. Although the sessile communities generally clustered separately from their planktonic counterparts, principal component analysis of carbon utilization profiles of the sessile communities showed different grouping between growth on chlorinated and nonchlorinated substrates. Analysis of the degradative community maintained on 2,4,6-trichlorobenzoic acid over an extended period further showed that adaptation to a new chemical environment is a rather slow process, since the substrate utilization profiles did not stabilize even after 12 months. These results demonstrate the flexibility in metabolic ability and community structure found in microbial communities. Received: 30 November 1998; Accepted: 19 May 1999     

Effects of Carbon Source, Carbon Concentration, and Chlorination on Growth Related Parameters of Heterotrophic Biofilm Bacteria

Abstract To investigate growth of heterotrophic biofilm bacteria, a model biofilm reactor was developed to simulate a drinking water distribution system. Controlled addition of three different carbon sources (amino acids, carbohydrates, and humics) at three different concentrations (500, 1,000, and 2,000 ppb carbon) in the presence and absence of chlorine were used in separate experiments. An additional experiment was run with a 1:1:2 mixture of the above carbon sources. Biofilm and effluent total and culturable cells in addition to total and dissolved organic carbon were measured in order to estimate specific growth rates (SGRs), observed yields, population densities, and bacterial carbon production rates. Bacterial carbon production rates (μg C/L day) were extremely high in the control biofilm communities (range = 295–1,738). Both growth rate and yield decreased with increasing carbon concentrations. Therefore, biofilm growth rates were zero-order with respect to the carbon concentrations used in these experiments. There was no correlation between growth rate and carbon concentration, but there was a significant negative correlation between growth rate and biofilm cell density (r=−0.637, p= 0.001 control and r=−0.57, p= 0.021 chlorinated biofilms). Growth efficiency was highest at the lowest carbon concentration (range = 12–4.5%, amino acids and humics respectively). Doubling times ranged from 2.3–15.4 days in the control biofilms and 1–12.3 days in the chlorinated biofilms. Growth rates were significantly higher in the presence of chlorine for the carbohydrates, humics, and mixed carbon sources (p= 0.004, < 0.0005, 0.013, respectively). The concept of r/K selection theory was used to explain the results with respect to specific growth rates and yields. Humic removal by the biofilm bacteria (78% and 56% for the control and chlorinated biofilms, respectively) was higher than previously reported literature values for planktonic bacteria. A number of control experiments indicated that filtration of drinking water was as effective as chlorination in controlling bacterial biofilm growth. Received: 26 March 1999; Accepted: 3 August 1999; Online Publication: 15 February 2000     

Analysis of Microbial Communities in a Landfill Leachate Polluted Aquifer using a New Method for Anaerobic Physiological Profiling and 16S rDNA Based Fingerprinting

Abstract Databases containing information regarding presence and activity of microbial communities will be very useful for determination of the potential for intrinsic bioremediation in landfill leachate polluted aquifers. Simple analyses such as community-level physiological profiling (CLPP) and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments yield large sets of data for inclusion into such databases. In this study we describe the development of a method for anaerobic CLPP, using commercially available Biolog plates. Incubation at the in situ temperature of the aquifer (10°C) for 28 days was optimal for obtaining a specific, reproducible physiological profile. Anaerobic incubation was essential for profiling anaerobic communities. The anaerobic cultivation-dependent CLPP method and cultivation-independent DGGE were applied to groundwater and sediment samples from the aquifer near the Coupépolder landfill in The Netherlands. A combination of computer-assisted CLPP and DGGE analysis of both groundwater and sediment samples yielded the best separating power for characterizing microbial communities in the aquifer. Communities in groundwater were significantly different from those in the corresponding sediment. Microbial communities present in subsamples from sediment cores usually were similar for the various sampling locations. Variation was observed for the heterogeneous sediment beneath the landfill. Both anaerobic CLPP and DGGE analysis clearly separated microbial communities from the polluted aquifer underneath the landfill from those in the less or not polluted aquifer downstream and upstream of the landfill. Received: 3 January 2000; Accepted: 21 March 2000; Online Publication: 28 August 2000     

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     

Phosphorus Limitation of Heterotrophic Biofilms from the Fraser River, British Columbia, and the Effect of Pulp Mill Effluent

Abstract Experiments were conducted to determine the limiting nutrient and the effect of pulp mill effluent (PME) on heterotrophic biofilms in the Fraser River, a northern, temperate river in British Columbia, Canada. Biofilms were cultured by irrigating flow cells with Fraser River water alone (control), or river water amended with glucose; ammonium; phosphate; glucose, ammonium, and phosphate combined; or 3% PME. Phosphorus increased bacterial growth in the biofilm, which was rapidly transferred to protists, and reduced the amount of extracellular polymeric substances accumulated by biofilm bacteria. This work demonstrates phosphorus limitation of biofilm bacteria in a major river system and emphasizes the importance of nutrients in industrial discharges. Received: 7 October 1997; Accepted: 8 January 1998     

Effects of Resources and Trophic Interactions on Freshwater Bacterioplankton Diversity

Abstract In a study of bacterioplankton in an oligotrophic lake in northern Wisconsin, a community fingerprinting technique, automated ribosomal intergenic spacer analysis (ARISA), was used to determine the effect of resources and trophic interactions on bacterioplankton diversity. Inorganic nitrogen and phosphorus (NP), carbon in the form of glucose (G) or dissolved organic matter extracted from peat (DOM), and carbon and NP in combination were added to two types of experimental systems. Ten-liter mesocosms contained all components of the original aquatic community except for large zooplankton. One-liter dilution cultures were prepared so that the effects of grazers and phytoplankton were removed. During a 3-day incubation, bacterial production showed the greatest response to the carbon plus NP treatment in both experimental systems, but bacterial diversity was strikingly different between them. In the mesocosms, the number of ARISA-PCR fragments averaged 41 per profile, whereas the dilution culture communities were highly reduced in complexity, dominated in most cases by a single PCR fragment. Further analysis of the mesocosm data suggested that whereas the NPDOM addition caused the greatest aggregate bacterial growth response, the addition of NP alone caused the largest shifts in community composition. These results suggest that the measurement of aggregate responses, such as bacterial production, alone in studies of freshwater bacterial communities may mask the effects of resources on bacterioplankton. Received: 24 January 2000; Accepted: 10 May 2000; Online Publication: 28 August 2000     

Protozoan grazing and its impact upon population dynamics in biofilm communities

AIMS: To determine the impact of protozoan grazing on the population dynamics of a multispecies bacterial biofilm community. METHODS AND RESULTS: Grazing by Acanthamoeba castellanii and the ciliate Colpoda maupasi upon biofilm and planktonic communities, composed of Klebsiella pneumoniae, Pseudomonas fluorescens and Staphylococcus epidermidis was investigated. Biofilms were formed using glass coverslips, held in a carousel device, as substrata for biofilm formation or in glass flow cells. The predatory effects of the amoeba were generally confined to the biofilm, where grazing rates corresponded to losses from the biofilm equivalent to ca 30,000 biofilm cells cm(-2) h(-1), with the amoeba becoming an integral part of the community. C. maupasi reduced the thickness of mature multispecies biofilms at steady-state from 500 to <200 microm. CONCLUSIONS: We report that the presence of the protozoa A. castellanii and C. maupasi markedly influence population dynamics within defined biofilm communities. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study dispels the popular opinion that biofilms are protected against predation by protozoa. A. castellanii clearly has the capacity to graze mixed biofilm communities and to become integrally associated with them, whereas the ciliate C. maupasi reduced biofilm thickness by up to 60%.     

Influence of Algal Biomass on Extracellular Enzyme Activity in River Biofilms

Abstract Relationships between biofilm structural components (algal and bacterial biomass) and the activities of some extracellular enzymes that contribute to the ability to degrade organic matter) were explored for six Atlantic and three Mediterranean streams and rivers. The biofilms in these fluvial systems accounted for a wide range of bacterial and algal biomass and colonized the most common benthic habitats. Ratio of bacteria/algae biomass was lower in Atlantic than in Mediterranean streams, but enzymatic activities (β-glucosidase, β-xylosidase, phosphatase) were in general greater in the Mediterranean stream biofilms. Climatic characteristics (especially temperature) may explain the differences in enzymatic activities between biofilms of similar structure but different flow regime. The ratio β-xylosidase: β-glucosidase was similar (around 0.5) for all streams and substrata considered, showing that there is a general higher utilization of cellobiosic than xylobiosic molecules in fluvial systems. In general, highly heterotrophic biofilms showed lower extracellular enzymatic activities than more autotrophic biofilms. Maximum enzymatic activity is achieved when the algal biomass is two- to threefold higher than the bacterial biomass. The relevance of algal biomass on the heterotrophic ability of biofilms may be related to the physical proximity between the two, but also to the high proportion of polymeric carbohydrates included in algal exudates and lysis products, whose use is enzyme-mediated. Received: January 2000; Accepted: April 2000; Online Publication: 18 July 2000     

<Emphasis Type="SmallCaps">d</Emphasis>-amino acids for the enhancement of a binary biocide cocktail consisting of THPS and EDDS against an SRB biofilm

Biofilms of sulfate reducing bacteria (SRB) are often responsible for Microbiologically Influenced Corrosion (MIC) that is a major problem in the oil and gas industry as well as water utilities and other industries. This work was inspired by recent reports that some d-amino acids may be useful in the control of microbial biofilms. A d-amino acid mixture with equimolar d-tyrosine, d-methionine, d-tryptophan and d-leucine was tested in this work for their enhancement of a biocide cocktail containing tetrakis (hydroxymethyl) phosphonium sulfate (THPS) and ethylenediamine-N,N’-disuccinic acid (EDDS). Desulfovibrio vulgaris (ATCC 7757) was cultured in ATCC 1249 medium. Its biofilm was grown on C1018 carbon steel coupons. Experimental results indicated that the triple biocide cocktail consisting of 30 ppm THPS, 500 ppm EDDS and 6.6 ppm d-amino acid mixture (with equimolar d-tyrosine, d-methionine, d-tryptophan and d-leucine) was far more effective than THPS and EDDS alone and their binary combination. The triple biocide cocktail effectively prevented SRB biofilm establishment and removed the established SRB biofilm. The d-amino acid mixture alone did not show significant effects in the two tasks even at 660 ppm.     

Fungal pyranose oxidases: occurrence, properties and biotechnical applications in carbohydrate chemistry

Pyranose oxidases are widespread among lignin-degrading white rot fungi and are localized in the hyphal periplasmic space. They are relatively large flavoproteins which oxidize a number of common monosaccharides on carbon-2 in the presence of oxygen to yield the corresponding 2-keto sugars and hydrogen peroxide. The preferred substrate of pyranose oxidases is d-glucose which is converted to 2-keto-d-glucose. While hydrogen peroxide is a cosubstrate in ligninolytic reactions, 2-keto-d-glucose is the key intermediate of a secondary metabolic pathway leading to the antibiotic cortalcerone. The finding that 2-keto-d-glucose can serve as an intermediate in an industrial process for the conversion of d-glucose into d-fructose has stimulated research on the use of pyranose oxidases in biotechnical applications. Unique catalytic potentials of pyranose oxidases have been discovered which make these enzymes efficient tools in carbohydrate chemistry. Converting common sugars and sugar derivatives with pyranose oxidases provides a pool of sugar-derived intermediates for the synthesis of a variety of rare sugars, fine chemicals and drugs. Received: 26 April 2000 / Received revision: 8 June 2000 / Accepted: 9 June 2000     

Unraveling assembly of stream biofilm communities

Microbial biofilms assemble from cells that attach to a surface, where they develop into matrix-enclosed communities. Mechanistic insights into community assembly are crucial to better understand the functioning of natural biofilms, which drive key ecosystem processes in numerous aquatic habitats. We studied the role of the suspended microbial community as the source of the biofilm community in three streams using terminal-restriction fragment length polymorphism and 454 pyrosequencing of the 16S ribosomal RNA (rRNA) and the 16S rRNA gene (as a measure for the active and the bulk community, respectively). Diversity was consistently lower in the biofilm communities than in the suspended stream water communities. We propose that the higher diversity in the suspended communities is supported by continuous inflow from various sources within the catchment. Community composition clearly differed between biofilms and suspended communities, whereas biofilm communities were similar in all three streams. This suggests that biofilm assembly did not simply reflect differences in the source communities, but that certain microbial groups from the source community proliferate in the biofilm. We compared the biofilm communities with random samples of the respective community suspended in the stream water. This analysis confirmed that stochastic dispersal from the source community was unlikely to shape the observed community composition of the biofilms, in support of species sorting as a major biofilm assembly mechanism. Bulk and active populations generated comparable patterns of community composition in the biofilms and the suspended communities, which suggests similar assembly controls on these populations.     

A comparative hierarchical analysis of bacterioplankton and biofilm metacommunity structure in an interconnected pond system

It is unknown whether bacterioplankton and biofilm communities are structured by the same ecological processes, and whether they influence each other through continuous dispersal (known as mass effects). Using a hierarchical sampling approach we compared the relative importance of ecological processes structuring the dominant fraction (relative abundance ≥0.1%) of bacterioplankton and biofilm communities from three microhabitats (open water, Nuphar and Phragmites sites) at within‐ and among‐pond scale in a set of 14 interconnected shallow ponds. Our results demonstrate that while bacterioplankton and biofilm communities are highly distinct, a similar hierarchy of ecological processes is acting on them. For both community types, most variation in community composition was determined by pond identity and environmental variables, with no effect of space. The highest β‐diversity within each community type was observed among ponds, while microhabitat type (Nuphar, Phragmites, open water) significantly influenced biofilm communities but not bacterioplankton. Mass effects among bacterioplankton and biofilm communities were not detected, as suggested by the absence of within‐site covariation of biofilm and bacterioplankton communities. Both biofilm and plankton communities were thus highly structured by environmental factors (i.e., species sorting), with among‐lake variation being more important than within‐lake variation, whereas dispersal limitation and mass effects were not observed.     

Enhancement of Sulphide Production in Anaerobic Packed Bed Bench-scale Biofilm Reactors by Sulphate Reducing Bacteria

Two biofilm reactors, using pumice stone and Poraver as biofilm supports, were run, based on the optimization of sulphide production using a factorial design. The maximum H₂S concentrations reached were 10 and 15 mm, respectively, both being appropriate for metal precipitation in effluents. The set-up of the pumice stone biofilm reactor is suitable for application in the mining area in the Bolivian Andean region, where this material is widely available. The use of specific primers for sulphate-reducing bacteria groups permits the identification of the sulphide-producing bacteria present in biofilms.     

Comparison of bacterial communities of biofilms formed on different membrane surfaces

Bacterial biofilm communities formed on different membrane surfaces were investigated based on 16S rRNA gene sequence analysis. The biofilm communities were distinct from those of mixed-liquor and consisted mainly of Beta- and Gammaproteobacteria. Sequences of Xathomonas and Aquabacterium were mostly retrieved from the biofilm samples rather than from the mixed liquor. Furthermore, statistical analyses demonstrated the importance of a physico-chemical property of membrane, surface roughness, in structuring the bacterial biofilm communities.     

Interspecific diversity reduces and functionally substitutes for intraspecific variation in biofilm communities

Diversity has a key role in the dynamics and resilience of communities and both interspecific (species) and intraspecific (genotypic) diversity can have important effects on community structure and function. However, a critical and unresolved question for understanding the ecology of a community is to what extent these two levels of diversity are functionally substitutable? Here we show, for a mixed-species biofilm community composed of Pseudomonas aeruginosa, P. protegens and Klebsiella pneumoniae, that increased interspecific diversity reduces and functionally substitutes for intraspecific diversity in mediating tolerance to stress. Biofilm populations generated high percentages of genotypic variants, which were largely absent in biofilm communities. Biofilms with either high intra- or interspecific diversity were more tolerant to SDS stress than biofilms with no or low diversity. Unexpectedly, genotypic variants decreased the tolerance of biofilm communities when experimentally introduced into the communities. For example, substituting P. protegens wild type with its genotypic variant within biofilm communities decreased SDS tolerance by twofold, apparently due to perturbation of interspecific interactions. A decrease in variant frequency was also observed when biofilm populations were exposed to cell-free effluents from another species, suggesting that extracellular factors have a role in selection against the appearance of intraspecific variants. This work demonstrates the functional substitution of inter- and intraspecific diversity for an emergent property of biofilms. It also provides a potential explanation for a long-standing paradox in microbiology, in which morphotypic variants are common in laboratory grown biofilm populations, but are rare in diverse, environmental biofilm communities.     

Differential uptake of fumarate by Candida utilis and Schizosaccharomyces pombe

The dicarboxylic acid fumarate is an important intermediate in cellular processes and also serves as a precursor for the commercial production of fine chemicals such as l-malate. Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. In this study we have shown that the yeast Candida utilis effectively degraded extracellular fumarate and l-malate, but glucose or other assimilable carbon sources repressed the transport and degradation of these dicarboxylic acids. The transport of both dicarboxylic acids was shown to be strongly inducible by either fumarate or l-malate while kinetic studies suggest that the two dicarboxylic acids are transported by the same transporter protein. In contrast, Schizosaccharomyces pombe effectively degraded extracellular l-malate, but not fumarate, in the presence of glucose or other assimilable carbon sources. The Sch. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of l-malate. Received: 15 March 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Proteomic analysis of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> in biofilm and planktonic growth mode

Recently, multidrug-resistant clinical isolates of Acinetobacter baumannii have been found to have a high capacity to form biofilm. It is well known that bacterial cells within biofilms are highly resistant to antibiotics, UV light, acid exposure, dehydration, and phagocytosis in comparison to their planktonic counterparts, which suggests that the cells in a biofilm have altered metabolic activity. To determine which proteins are up-regulated in A. baumannii biofilm cells, we performed a proteomic analysis. A clinical isolate of A. baumannii 1656-2, which was characterized to have a high biofilm forming ability, was cultivated under biofilm and planktonic conditions. Outer membrane enriched A. baumannii 1656-2 proteins were separated by two-dimensional (2-D) gel electrophoresis and the differentially expressed proteins were identified by MALDI-TOF mass spectrometry. The proteins up-regulated or expressed only in biofilm cells of A. baumannii are categorized as follows: (i) proteins processing environmental information such as the outer membrane receptor protein involved in mostly Fe transport, a sensor histidine kinase/response regulator, and diguanylate cyclase (PAS-GGEDF-EAL domain); (ii) proteins involved in metabolism such as NAD-linked malate dehydrogenase, nucleoside-diphosphate sugar epimerase, putative GalE, ProFAR isomerase, and N-acetylmuramoyl-l-alanine amidase; (iii) bacterial antibiotic resistance related proteins; and (iv) proteins related to gene repair such as exodeoxyribonuclease III and GidA. This proteomic analysis provides a fundamental platform for further studies to reveal the role of biofilm in the persistence and tolerance of A. baumannii.     

Enrichment versus biofilm culture: a functional and phylogenetic comparison of polycyclic aromatic hydrocarbon-degrading microbial communities

The effect that culture methods have on the diversity of degradative microbial communities is not well understood. We compared conventional batch enrichment with a biofilm culture method for the isolation of polycyclic aromatic hydrocarbon (PAH)-degrading microbial communities from a PAH-contaminated soil. The two methods were assessed by comparing: (i) the diversity of culturable bacteria; (ii) the diversity of PAH-catabolic genes in isolated bacteria; (iii) the inter- and intraspecific diversity of active PAH-catabolic gene classes; (iv) the diversity of bacteria present in 16S rRNA gene libraries generated from RNA extracted from the two communities and soil; and (v) the estimated diversity of active bacteria in the soil and culture systems. Single-strand conformation polymorphism analysis showed that the biofilm culture yielded 36 bacterial and two fungal species compared with 12 bacterial species from the enrichment culture. Application of accumulation and non-parametric estimators to clone libraries generated from 16S rRNA confirmed that the biofilm community contained greater diversity. Sequencing of clones showed that only species from the Proteobacteria were active in the enrichment culture, and that these species were expressing an identical nahAc-like naphthalene dioxygenase. 16S rRNA clones generated from the biofilm community indicated that species from the Cytophaga/Flavobacterium, high G+C bacteria and Proteobacteria were active at the time of sampling, expressing cndA-, nahAc- and phnAc-like naphthalene dioxygenases. The diversity of active species in the biofilm culture system closely matched that in the PAH-contaminated source soil. The results of this study showed that biofilm culture methods are more appropriate for the study of community-level interactions in PAH-degrading microbial communities. The study also indicated that cultivation of microbial communities on solid media might be the primary source of bias in the recovery of diverse species.