Impact of flow velocity on the dynamic behaviour of biofilm bacteria

The impact of flow velocity (FV) on the growth dynamics of biofilms and bulk water heterotrophic plate count (HPC) bacteria in drinking water distribution systems was quantified and modeled by combining a logistic growth model with mass balance equations. The dynamic variations in the specific growth and release rates of biofilm bacteria were also quantified. The experimental results showed that the maximum biofilm biomass did not change when flow velocity was increased from 20 to 40 cm s(-1), but was significantly affected when flow velocity was further increased to 60 cm s(-1). Although the concentration of biofilm bacteria was substantially reduced by the higher shear stress, the concentration of bacteria in the bulk fluid was slightly increased. From this it is estimated that the specific growth rate and specific release rate of biofilm bacteria had doubled. The specific release (detachment) rate was dependent on the specific growth rate of the biofilm bacteria.     

‘Should I stay or should I go?’ Bacterial attachment vs biofilm formation on surface-modified membranes

A number of techniques are used for testing the anti-biofouling activity of surfaces, yet the correlation between different results is often questionable. In this report, the correlation between initial bacterial deposition (fast tests, reported previously) and biofilm growth (much slower tests) was analyzed on a pristine and a surface-modified reverse osmosis membrane ESPA-1. The membrane was modified with grafted hydrophilic polymers bearing negatively charged, positively charged and zwitter-ionic moieties. Using three different bacterial strains it was found that there was no general correlation between the initial bacterial deposition rates and biofilm growth on surfaces, the reasons being different for each modified surface. For the negatively charged surface the slowest deposition due to the charge repulsion was eventually succeeded by the largest biofilm growth, probably due to secretion of extracellular polymeric substances (EPS) that mediated a strong attachment. For the positively charged surface, short-term charge attraction by quaternary amine groups led to the fastest deposition, but could be eventually overridden by their antimicrobial activity, resulting in non-consistent results where in some cases a lower biofilm formation rate was observed. The results indicate that initial deposition rates have to be used and interpreted with great care, when used for assessing the anti-biofouling activity of surfaces. However, for a weakly interacting ‘low-fouling’ zwitter-ionic surface, the positive correlation between initial cell deposition and biofilm growth, especially under flow, suggests that for this type of coating initial deposition tests may be fairly indicative of anti-biofouling potential.     

Shear rate moderates community diversity in freshwater biofilms

The development of freshwater multispecies biofilms at solid-liquid interfaces occurs both in quiescent waters and under conditions of high shear rates. However, the influence of hydrodynamic shear rates on bacterial biofilm diversity is poorly understood. We hypothesized that different shear rates would significantly influence biofilm diversity and alter the relative proportions of coaggregating and autoaggregating community isolates. In order to study this hypothesis, freshwater biofilms were developed at five shear rates (<0.1 to 305 S(-1)) in a rotating concentric cylinder reactor fed with untreated potable water. Eubacterial diversity was assessed by denaturing gradient gel electrophoresis (DGGE) and culturing on R2A agar. Fifty morphologically distinct biofilm strains and 16 planktonic strains were isolated by culturing and identified by partial 16S rRNA gene sequencing, and their relatedness was determined by the construction of a neighbor-joining phylogenetic tree. Phylogenetic and DGGE analyses showed an inverse relationship between shear rate and bacterial diversity. An in vitro aggregation assay was used to assess the relative proportions of coaggregating and autoaggregating species from each biofilm. The highest proportion of autoaggregating bacteria was present at high shear rates (198 to 305 S(-1)). The intermediate shear rate (122 S(-1)) selected for the highest proportion of coaggregating bacteria (47%, or 17 of a possible 36 coaggregation interactions). Under static conditions (<0.1 S(-1)), 41 (33%) of a possible 125 coaggregation interactions were positive. Few coaggregation (3.3%) or autoaggregation (25%) interactions occurred between the 16 planktonic strains. In conclusion, these data show that shear rates affect biofilm diversity as well as the relative proportions of aggregating bacteria.     

The influence of flow cell geometry related shear stresses on the distribution,structure and susceptibility of Pseudomonas aeruginosa 01 biofilms

The effects of non-uniform hydrodynamic conditions resulting from flow cell geometry (square and rectangular cross-section) on Pseudomonas aeruginosa 01 (PAO1) biofilm formation, location, and structure were investigated for nominally similar flow conditions using a combination of confocal scanning laser microscope (CSLM) and computational fluid dynamics (CFD). The thickness and surface coverage of PAO1 biofilms were observed to vary depending on the location in the flow cell and thus also the local wall shear stress. The biofilm structure in a 5:1 (width to height) aspect ratio rectangular flow cell was observed to consist mainly of a layer of bacterial cells with thicker biofilm formation observed in the flow cell corners. For square cross-section (1:1 aspect ratio) flow cells, generally thicker and more uniform surface coverage biofilms were observed. Mushroom shaped structures with hollow centers and wall breaks, indicative of ‘seeding’ dispersal structures, were found exclusively in the square cross-section tubes. Exposure of PAO1 biofilms grown in the flow cells to gentamicin revealed a difference in susceptibility. Biofilms grown in the rectangular flow cell overall exhibited a greater susceptibility to gentamicin compared to those grown in square flow cells. However, even within a given flow cell, differences in susceptibility were observed depending on location. This study demonstrates that the spanwise shear stress distribution within the flow cells has an important impact on the location of colonization and structure of the resultant biofilm. These differences in biofilm structure have a significant impact on the susceptibility of the biofilms grown within flow channels. The impact of flow modification due to flow cell geometry should be considered when designing flow cells for laboratory investigation of bacterial biofilms.     

A new coupon design for simultaneous analysis of in situ microbial biofilm formation and community structure in drinking water distribution systems

This study presents a new coupon sampling device that can be inserted directly into the pipes within water distribution systems (WDS), maintaining representative near wall pipe flow conditions and enabling simultaneous microscopy and DNA-based analysis of biofilms formed in situ. To evaluate this sampling device, fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate changes in biofilms on replicate coupons within a non-sterile pilot-scale WDS. FISH analysis demonstrated increases in bacterial biofilm coverage of the coupon surface over time, while the DGGE analysis showed the development of increasingly complex biofilm communities, with time-specific clustering of these communities. This coupon design offers improvements over existing biofilm sampling devices in that it enables simultaneous quantitative and qualitative compositional characterization of biofilm assemblages formed within a WDS, while importantly maintaining fully representative near wall pipe flow conditions. Hence, it provides a practical approach that can be used to capture the interactions between biofilm formation and changing abiotic conditions, boundary shear stress, and turbulent driven exchange within WDS.     

Bacterial species dominance within a binary culture biofilm

Studies with two species of bacteria, Pseudomonas putida and Hyphomicrobium sp. strain ZV620, were carried out to evaluate the overall net rate of accumulation of biofilm, the biofilm species composition, and individual species shear-related removal rates. Bacterial cells of either or both species were deposited onto glass or biofilm surfaces to initiate multispecies biofilms. Subsequent biofilm development was carried out under known conditions of nutrient concentration and laminar flow. Establishment of a depositing organism in a biofilm composed of another species was found to be a function of the relative growth rates of the bacterial species. In the case of simultaneous species deposition and subsequent binary culture development, the faster-growing organisms rapidly became the dominant biofilm species, but the slower-growing organisms remained established within the biofilm and continued to increase in numbers over time. The results also indicated that the rate of cell removal by fluid shear for a species was a function of biofilm cell number only if the species concentration was uniform with depth; in essence, only the upper layers of the biofilm were sheared off.     

Bacterial species dominance within a binary culture biofilm.

Studies with two species of bacteria, Pseudomonas putida and Hyphomicrobium sp. strain ZV620, were carried out to evaluate the overall net rate of accumulation of biofilm, the biofilm species composition, and individual species shear-related removal rates. Bacterial cells of either or both species were deposited onto glass or biofilm surfaces to initiate multispecies biofilms. Subsequent biofilm development was carried out under known conditions of nutrient concentration and laminar flow. Establishment of a depositing organism in a biofilm composed of another species was found to be a function of the relative growth rates of the bacterial species. In the case of simultaneous species deposition and subsequent binary culture development, the faster-growing organisms rapidly became the dominant biofilm species, but the slower-growing organisms remained established within the biofilm and continued to increase in numbers over time. The results also indicated that the rate of cell removal by fluid shear for a species was a function of biofilm cell number only if the species concentration was uniform with depth; in essence, only the upper layers of the biofilm were sheared off.     

Bacterial repopulation of drinking water pipe walls after chlorination

The short-term kinetics of bacterial repopulation were evaluated after chlorination of high-density polyethylene (HDPE) colonized with drinking water biofilms and compared with bare HDPE surfaces. The effect of chlorination was partial as a residual biofilm persisted and was time-limited as repopulation occurred immediately after water resupply. The total number of bacteria reached the same levels on both the bare and chlorinated biofilm-fouled HDPE after a seven-day exposure to drinking water. Due to the presence of a residual biofilm, the hydrophobicity of chlorinated biofilm-fouled surface exhibited much lower adhesion forces (2.1 nN) compared to bare surfaces (8.9 nN). This could explain the rapid repopulation after chlorination, with a twofold faster bacterial accumulation rate on the bare HDPE surface. γ-Proteobacteria dominated the early stages of repopulation of both surfaces and a shift in the dominance occurred over the colonization time. Such observations define a timescale for cleaning frequency in industrial environments and guidelines for a rinsing procedure using drinking water.     

Characterisation of the Physical Composition and Microbial Community Structure of Biofilms within a Model Full-Scale Drinking Water Distribution System

Within drinking water distribution systems (DWDS), microorganisms form multi-species biofilms on internal pipe surfaces. A matrix of extracellular polymeric substances (EPS) is produced by the attached community and provides structure and stability for the biofilm. If the EPS adhesive strength deteriorates or is overcome by external shear forces, biofilm is mobilised into the water potentially leading to degradation of water quality. However, little is known about the EPS within DWDS biofilms or how this is influenced by community composition or environmental parameters, because of the complications in obtaining biofilm samples and the difficulties in analysing EPS. Additionally, although biofilms may contain various microbial groups, research commonly focuses solely upon bacteria. This research applies an EPS analysis method based upon fluorescent confocal laser scanning microscopy (CLSM) in combination with digital image analysis (DIA), to concurrently characterize cells and EPS (carbohydrates and proteins) within drinking water biofilms from a full-scale DWDS experimental pipe loop facility with representative hydraulic conditions. Application of the EPS analysis method, alongside DNA fingerprinting of bacterial, archaeal and fungal communities, was demonstrated for biofilms sampled from different positions around the pipeline, after 28 days growth within the DWDS experimental facility. The volume of EPS was 4.9 times greater than that of the cells within biofilms, with carbohydrates present as the dominant component. Additionally, the greatest proportion of EPS was located above that of the cells. Fungi and archaea were established as important components of the biofilm community, although bacteria were more diverse. Moreover, biofilms from different positions were similar with respect to community structure and the quantity, composition and three-dimensional distribution of cells and EPS, indicating that active colonisation of the pipe wall is an important driver in material accumulation within the DWDS.     

Novel experimental study of receptor-mediated bacterial adhesion under the influence of fluid shear

Dynamic adhesion of cells to surfaces is a vital step in a variety of biochemical and physiological phenomena. Bacterial adhesion is responsible not only for problems associated with biofouling and biofilm formation in the biochemical industry but also in the initiation of certain infectious diseases. In this study, we report the effect of critical parameters, such as receptor and ligand densities and shear rate, on receptor-mediated dynamic bacterial adhesion. Adhesion of a pathogenic strain of Staphylococcus aureus to immobilized collagen was studied. The receptor density on the cell surface was varied by harvesting cells at different growth times and was quantified using flow cytometry. Dynamic adhesion experiments were conducted over a range of physiologically relevant shear rates (50 to 1500 s(-1)) using a parallel-plate flow chamber. Video microscopy coupled with digital image processing was employed to quantify adhesion. A semiquantitative comparison between experimental results and theoretical data obtained using a previously proposed mathematical model was also performed. The results suggest that dynamic adhesion is dependent on receptor density and shear rate, but independent of ligand density. This report demonstrates the feasibility of using bacteria to study fundamental aspects of receptor-mediated dynamic adhesion.     

Direct upstream motility in Escherichia coli

We provide an experimental demonstration of positive rheotaxis (rapid and continuous upstream motility) in wild-type Escherichia coli freely swimming over a surface. This hydrodynamic phenomenon is dominant below a critical shear rate and robust against Brownian motion and cell tumbling. We deduce that individual bacteria entering a flow system can rapidly migrate upstream (>20 μm/s) much faster than a gradually advancing biofilm. Given a bacterial population with a distribution of sizes and swim speeds, local shear rate near the surface determines the dominant hydrodynamic mode for motility, i.e., circular or random trajectories for low shear rates, positive rheotaxis for moderate flow, and sideways swimming at higher shear rates. Faster swimmers can move upstream more rapidly and at higher shear rates, as expected. Interestingly, we also find on average that both swim speed and upstream motility are independent of cell aspect ratio.     

Bacterial community in the biofilm of granular activated carbon (GAC) PreBiofilter in bench-scale pilot plants for surface water pretreatment

Biofilters of granular activated carbon (GAC) are responsible for the removal of organic matters in drinking water treatments. PreBiofilters, which operate as the first unit in a surface water treatment train, are a cost-effective pretreatment for conventional surface water treatment and provide more consistent downstream water quality. This study investigated bacterial communities from the samples of raw surface water, biofilm on the PreBiofilter, and filtrates for surface water pretreatment. A bench-scale pilot plant of PreBiofilter was constructed to pretreat surface water from the Canoochee River, GA, USA. PreBiofilter exhibited a significant reduction of total organic carbon and dissolved organic carbon. The evenness and Shannon diversity of bacterial operational taxonomic units (OTUs) were significantly higher on the biofilm of PreBiofilter than in raw water and filtrates. Similar bacteria communities were observed in the raw water and filtrates using relative abundance of bacterial OTUs. However, the bacterial communities in the filtrates became relatively similar to those in the biofilm using presence/absence of bacterial OTUs. GAC biofilm or raw water and filtrates greatly contributed to the abundance of bacteria; whereas, bacteria sheared from colonized biofilm and entered filtrates. Evenly distributed, diverse and unique bacteria in the biofilm played an important role to remove organic matters from surface water for conventional surface water pretreatment.     

Shear Rate Moderates Community Diversity in Freshwater Biofilms

The development of freshwater multispecies biofilms at solid-liquid interfaces occurs both in quiescent waters and under conditions of high shear rates. However, the influence of hydrodynamic shear rates on bacterial biofilm diversity is poorly understood. We hypothesized that different shear rates would significantly influence biofilm diversity and alter the relative proportions of coaggregating and autoaggregating community isolates. In order to study this hypothesis, freshwater biofilms were developed at five shear rates (<0.1 to 305 S⁻¹) in a rotating concentric cylinder reactor fed with untreated potable water. Eubacterial diversity was assessed by denaturing gradient gel electrophoresis (DGGE) and culturing on R2A agar. Fifty morphologically distinct biofilm strains and 16 planktonic strains were isolated by culturing and identified by partial 16S rRNA gene sequencing, and their relatedness was determined by the construction of a neighbor-joining phylogenetic tree. Phylogenetic and DGGE analyses showed an inverse relationship between shear rate and bacterial diversity. An in vitro aggregation assay was used to assess the relative proportions of coaggregating and autoaggregating species from each biofilm. The highest proportion of autoaggregating bacteria was present at high shear rates (198 to 305 S⁻¹). The intermediate shear rate (122 S⁻¹) selected for the highest proportion of coaggregating bacteria (47%, or 17 of a possible 36 coaggregation interactions). Under static conditions (<0.1 S⁻¹), 41 (33%) of a possible 125 coaggregation interactions were positive. Few coaggregation (3.3%) or autoaggregation (25%) interactions occurred between the 16 planktonic strains. In conclusion, these data show that shear rates affect biofilm diversity as well as the relative proportions of aggregating bacteria.     

Interaction of chlorine concentration and shear stress on chlorine consumption, biofilm growth rate and particle number

Eleven test runs (including two replicates) were carried out to explore the interaction of shear stress and chlorine concentration on the growth of heterotrophic microorganisms. Experimental results revealed that influent chlorine concentration and shear stress had no interaction on biofilm formation. Biofilm bacterial numbers decreased with the increase of influent chlorine concentration. Increasing the shear stress up to a specific level could significantly reduce the potential of biofilm formation. A strong interaction on bacterial quality or chlorine consumption rate of bulk water existed. With non-chlorinated and lower chlorinated conditions, the specific growth rate of biofilm increased with the increase of shear stress. However, an inverse relation occurred at higher chlorine conditions. No significant interaction of chlorine concentration and shear stress existed for particle numbers with 2-5, 5-15, 50-100 and >100 microm diameters. However, a significant interaction existed on particle numbers of 15-25 and 25-50 microm diameters.     

Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation

The influence of type 1 fimbriae, mannose-sensitive structures, on biofilm development and maturation has been examined by the use of three isogenic Escherichia coli K12 strains: wild type, fimbriated, and non-fimbriated. Experiments with the three strains were done in minimal medium or Luria–Bertani broth supplemented with different concentrations of d-mannose. The investigation consisted of: (1) characterizing the bacterial surface of the three strains with respect to hydrophilicity and surface charge, (2) investigating the effect of type 1 fimbriae on bacterial adhesion rate and reversibility of initial adhesion on glass surfaces, and (3) verifying the role of type 1 fimbriae and exopolysaccharides (EPS) in biofilm maturation. The results suggest that type 1 fimbriae are not required for the initial bacterial adhesion on glass surfaces as the non-fimbriated cells had higher adhesion rates and irreversible deposition. Type 1 fimbriae, however, are critical for subsequent biofilm development. It was hypothesized that in the biofilm maturation step, the cells synthesize mannose-rich EPS, which functions as a ‘conditioning film’ that can be recognized by the type 1 fimbriae.     

Influence of biofilms on iron and manganese deposition in drinking water distribution systems

Although health risk due to discoloured water is minimal, such water continues to be the source of one of the major complaints received by most water utilities in Australia. Elevated levels of iron (Fe) and/or manganese (Mn) in bulk water are associated with discoloured water incidents. The accumulation of these two elements in distribution systems is believed to be one of the main causes for such elevated levels. An investigation into the contribution of pipe wall biofilms towards Fe and Mn deposition, and discoloured water events is reported in this study. Eight laboratory-scale reactors were operated to test four different conditions in duplicate. Four reactors were exposed to low Fe (0.05 mg l(-1)) and Mn (0.02 mg l(-1)) concentrations and the remaining four were exposed to a higher (0.3 and 0.4 mg l(-1) for Fe and Mn, respectively) concentration. Two of the four reactors which received low and high Fe and Mn concentrations were chlorinated (3.0 mg l(-1) of chlorine). The biological activity (measured in terms of ATP) on the glass rings in these reactors was very low (～1.5 ng cm(-2) ring). Higher concentrations of Fe and Mn in bulk water and active biofilms resulted in increased deposition of Fe and Mn on the glass rings. Moreover, with an increase in biological activity, an increase in Fe and Mn deposition was observed. The observations in the laboratory-scale experiments were in line with the results of field observations that were carried out using biofilm monitors. The field data additionally demonstrated the effect of seasons, where increased biofilm activities observed on pipe wall biofilms during late summer and early autumn were found to be associated with increased deposition of Fe and Mn. In contrast, during the cooler months, biofilm activities were a magnitude lower and the deposited metal concentrations were also significantly less (ie a drop of 68% for Fe and 86% for Mn). Based on the laboratory-scale investigations, detachment of pipe wall biofilms due to cell death or flow dynamics could release the entrapped Fe and Mn into the bulk water, which could lead to a discoloured water event. Hence, managing biofilm growth on drinking water pipelines should be considered by water utilities to minimize accumulation of Fe and Mn in distribution networks.     

Impacts of hydrodynamic conditions and microscale surface roughness on the critical shear stress to develop and thickness of early-stage Pseudomonas putida biofilms

Biofilms can increase pathogenic contamination of drinking water, cause biofilm-related diseases, alter the sediment erosion rate, and degrade contaminants in wastewater. Compared with mature biofilms, biofilms in the early-stage have been shown to be more susceptible to antimicrobials and easier to remove. Mechanistic understanding of physical factors controlling early-stage biofilm growth is critical to predict and control biofilm development, yet such understanding is currently incomplete. Here, we reveal the impacts of hydrodynamic conditions and microscale surface roughness on the development of early-stage Pseudomonas putida biofilm through a combination of microfluidic experiments, numerical simulations, and fluid mechanics theories. We demonstrate that early-stage biofilm growth is suppressed under high flow conditions and that the local velocity for early-stage P. putida biofilms (growth time < 14 h) to develop is about 50 μm/s, which is similar to P. putida's swimming speed. We further illustrate that microscale surface roughness promotes the growth of early-stage biofilms by increasing the area of the low-flow region. Furthermore, we show that the critical average shear stress, above which early-stage biofilms cease to form, is 0.9 Pa for rough surfaces, three times as large as the value for flat or smooth surfaces (0.3 Pa). The important control of flow conditions and microscale surface roughness on early-stage biofilm development, characterized in this study, will facilitate future predictions and managements of early-stage P. putida biofilm development on the surfaces of drinking water pipelines, bioreactors, and sediments in aquatic environments.     

Simultaneous nitrification and denitrification using an autotrophic membrane-immobilized biofilm reactor

AIMS: To develop a laboratory-scale autotrophic membrane-immobilized biofilm reactor to remove nitrogen from drinking water. METHODS AND RESULTS: A polyvinyl alcohol (PVA) immobilized biofilm, attached to the surface of a silicone tube, was used as the basis of a bioreactor for simultaneous nitrification and denitrification of water. The bioreactor was aerated with air to supply oxygen for nitrification. Pure hydrogen was supplied to the silicone tube and diffused through the membrane wall to feed the biofilm for autotrophic denitrification. The bioreactor was effective for the simultaneous nitrification and denitrification of water after a short period of acclimation, while the biofilm exhibited good resistance to the inhibition of denitrification by dissolved oxygen; the denitrification rate decreased by only 8% as the dissolved oxygen increased from 2 mg l(-1) to saturation. CONCLUSIONS: By using PVA crosslinked with sodium nitrate to entrap nitrifying and denitrifying sludge on the surface of a silicone tube, a novel bioreactor for simultaneous nitrification and denitrification was developed. In addition to performing as an immobilizing agent to strengthen the biofilm, PVA protected the denitrifying microorganisms to reduce the inhibition by dissolved oxygen under aerobic condition. Therefore, nitrification and denitrification occurred simultaneously within the biofilm. Furthermore, the immobilization technique shortened the acclimation period of the bioreactor. SIGNIFICANCE AND IMPACT OF THE STUDY: The described space saving and simple to operate bioreactor for nitrogen removal performed autotrophic denitrification to solve the problem of residual carbon in heterotrophic denitrification, and thus is suitable for removing nitrogen from drinking water.     

Impact of flow velocity on the dynamic behaviour of biofilm bacteria

Abstract

The impact of flow velocity (FV) on the growth dynamics of biofilms and bulk water heterotrophic plate count (HPC) bacteria in drinking water distribution systems was quantified and modeled by combining a logistic growth model with mass balance equations. The dynamic variations in the specific growth and release rates of biofilm bacteria were also quantified. The experimental results showed that the maximum biofilm biomass did not change when flow velocity was increased from 20 to 40 cm s^?1, but was significantly affected when flow velocity was further increased to 60 cm s^?1. Although the concentration of biofilm bacteria was substantially reduced by the higher shear stress, the concentration of bacteria in the bulk fluid was slightly increased. From this it is estimated that the specific growth rate and specific release rate of biofilm bacteria had doubled. The specific release (detachment) rate was dependent on the specific growth rate of the biofilm bacteria.     

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