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PubMed2004

Effects of substrate loading rate on biofilm structure.

The effects of substrate surface loading rate on biofilm growth and structure were investigated by chemical, biochemical and microscopic methods. Three tubular reactors were operated at equal C:N ratio of 0.1, with substrate loading rates of 1.2, 0.6 and 0.3g-C/m(2)/day. Substrate loading positively influenced the biofilm growth rate. Denser biofilms with lower porosities were formed at higher substrate loading. Slowly growing biofilms having porous structures were found to have higher specific activities. Nitrification was suppressed under the higher substrate loading conditions even at the equal C:N ratio of 0.1, thus proving that the spatial competition between nitrifiers and heterotrophs as one limiting criteria for stable nitrification. The spatial organization of the ammonia oxidizers was biofilm structure related. The strain variability of ammonia oxidizers was substrate loading dependent. These findings suggest that substrate loading is a key parameter in determining biofilm structure and function.

Ammonia↗
PubMedDate not supplied

Modeling the development of biofilm density including active bacteria, inert biomass, and extracellular polymeric substances.

We present the unified multi-component cellular automaton (UMCCA) model, which predicts quantitatively the development of the biofilm's composite density for three biofilm components: active bacteria, inert or dead biomass, and extracellular polymeric substances. The model also describes the concentrations of three soluble organic components (soluble substrate and two types of soluble microbial products) and oxygen. The UMCCA model is a hybrid discrete-differential mathematical model and introduces the novel feature of biofilm consolidation. Our hypothesis is that the fluid over the biofilm creates pressures and vibrations that cause the biofilm to consolidate, or pack itself to a higher density over time. Each biofilm compartment in the model output consolidates to a different degree that depends on the age of its biomass. The UMCCA model also adds a cellular automaton algorithm that identifies the path of least resistance and directly moves excess biomass along that path, thereby ensuring that the excess biomass is distributed efficiently. A companion paper illustrates the trends that the UMCCA model is able to represent and shows a comparison with experimental results.

Algorithms↗
PubMed2004

Evaluation of disinfectant efficacy against biofilm and suspended bacteria in a laboratory swimming pool model.

Laboratory reactor systems designed to model specific environments enable researchers to explore environmental dynamics in a more controlled manner. This paper describes the design and operation of a reactor system built to model a swimming pool in the laboratory. The model included relevant engineering parameters such as filter loading and turn-overs per day. The water chemistry in the system's bulk water was balanced according to standard recommendations and the system was challenged with a bacterial load and synthetic bather insult, formulated to represent urine and perspiration. The laboratory model was then used to evaluate the efficacy of six chemical treatments against biofilm and planktonic bacteria. Results showed that the biofilm was able to accumulate on coupons and in the filter systems of reactors treated with either 1-3 mg/L free chlorine or 10 mg/L polyhexamethylene biguanide (PHMB). All the treatments tested resulted in at least a 4 log reduction in biofilm density when compared to the control, but shock treatments were the most effective at controlling biofilm accumulation. A once weekly shock dose of 10 mg/L free chlorine resulted in the greatest log reduction in biofilm density. The research demonstrated the importance of studying a biofilm in addition to the planktonic bacteria to assess the microbial dynamics that exist in a swimming pool model.

Bacteria↗
PubMed2004

Microbiology, chemistry and biofilm development in a pilot drinking water distribution system with copper and plastic pipes.

We studied the changes in water quality and formation of biofilms occurring in a pilot-scale water distribution system with two generally used pipe materials: copper and plastic (polyethylene, PE). The formation of biofilms with time was analysed as the number of total bacteria, heterotrophic plate counts and the concentration of ATP in biofilms. At the end of the experiment (after 308 days), microbial community structure, viable biomass and gram-negative bacterial biomass were analysed via lipid biomarkers (phospholipid fatty acids and lipopolysaccharide 3-hydroxy fatty acids), and the numbers of virus-like particles and total bacteria were enumerated by SYBR Green I staining. The formation of biofilm was slower in copper pipes than in the PE pipes, but after 200 days there was no difference in microbial numbers between the pipe materials. Copper ion led to lower microbial numbers in water during the first 200 days, but thereafter there were no differences between the two pipe materials. The number of virus-like particles was lower in biofilms and in outlet water from the copper pipes than PE pipes. Pipe material influenced also the microbial and gram-negative bacterial community structure in biofilms and water.

Biofilms↗
PubMed2004

Occurrence of nitrifiers and diversity of ammonia-oxidizing bacteria in developing drinking water biofilms.

We studied the population dynamics of nitrifying bacteria during the development of biofilms up to 233 or 280 days on polyvinylchloride pipes connected to two full-scale drinking water distribution networks supplying processed and chloraminated surface water. The numbers of nitrifiers in biofilms were enumerated at intervals of 10-64 days by the most probable number (MPN) method at waterworks and at several study sites in distribution network areas. The numbers of nitrifiers increased towards the distal sites. The highest detected MPN counts of ammonia-oxidizing bacteria (AOB) for study areas 1 and 7 were 500 MPN cm(-2) and 1.0 x 10(6) MPN cm(-2), and those of nitrite-oxidizing bacteria (NOB) 96 MPN cm(-2) and 2.2 x 10(3) MPN cm(-2), respectively. The diversity of AOB was determined by PCR amplifying, cloning and sequencing the partial ammonia monooxygenase (amoA) gene of selected biofilm samples presenting different biofilm ages. The PCR primers used, A189 and A682, also amplified a fragment of particulate methane monooxygenase (pmoA) gene of methane-oxidizing bacteria. The majority of biofilm clones (24 out of 30 studied) contained Nitrosomonas amoA-like sequences. There were only two pmoA-like sequences of Type I methanotrophs, and four sequences positioned in amoA/pmoA sequence groups of uncultured bacteria. From both study area very similar or even completely identical Nitrosomonas amoA-like sequences were obtained despite of high difference in AOB numbers. The results show that the conditions in newly formed biofilms in drinking water distribution systems favor the growth of Nitrosomonas-type AOB.

Ammonia↗
PubMed2004

Analysis of bacterial diversity in river biofilms using 16S rDNA PCR-DGGE: methodological settings and fingerprints interpretation.

Reliability of bacterial diversity assessment using polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA fragments was evaluated for a particular complex microbial assemblage: river epilithic biofilm. By comparing 3 routine protocols on replicates of one river biofilm sample, we found that common DNA extraction procedures gave comparable diversity (from 28.0 to 30.7 bands detected) and community composition (> 75% of homology) despite differences in the total amount of extracted DNA (from 0.9 to 4.2 microg). Therefore methodological improvements only concerned electrophoretic separation of DNA fragments (range of denaturing gradient from 35% to 70% and migration time=18h) and standardisation of DNA amounts used (PCR-template=50 ng, gel loading=700 ng). Using such a standardised methodology we found a good reproducibility of all steps of the procedure. When an Escherichia coli strain was introduced as a contaminant in a biofilm sample, we were able to recover ribotypes from the strain. As concerns fields sampling, a satisfactory repeatability of banding patterns from neighbouring pebbles (sampling point) allowed discriminating between the biofilm intrasite variability (various points from a cross-profile). These trials confirmed that PCR-DGGE is suitable to assess a reliable genetic fingerprint of epilithic biofilms in the river. Phylogenetic analysis of 40 partial sequences of 16S rDNA from DGGE gels of two sets of river biofilms samples proved evidences for the retrieval of DNA fragments related to phototroph Eukarya. However, in both cases plastidial 16S rDNA represented less than 25% of the analysed operational taxonomic units. Taking into account that Cyanobacteria, as members of the Bacteria, were also detected, sequence analysis of relevant bands from the pattern is required to target "bacteria", i.e. the functional group of prokaryotic microorganisms to which one commonly refers as a key component in sustaining the nutrient turnover.

Bacteria↗
PubMed2005

Modeling biofilm and floc diffusion processes based on analytical solution of reaction-diffusion equations.

Biofilm modeling is often considered as a complex mathematical subject. This paper evaluates simple equations to describe the basic processes in a biofilm system with the main aim to show several interesting applications. To avoid mathematical complexity the simulations are carried out in a simple spreadsheet. Frequently, only the solution for zero-order reaction kinetics of the reaction-diffusion equation is used (better known as half-order kinetics). A weighted average of the analytical solutions for zero- and first-order reactions is proposed as basic and useful model to describe steady-state (in biofilm composition) biofilm reactors. This approach is compared with several modeling approaches, such as the simple solution for zero-order reaction and more complex ones (i) direct numerical solution for the diffusion equations, (ii) 1-D AQUASIM and (iii) 2-D modeling. The systems evaluated are single and multiple species biofilms. It is shown that for describing conversions in biofilm reactors, the zero-order solution is generally sufficient; however, for design purposes large deviations of the correct solution can occur. Additionally, the role of diffusion in flocculated and granular sludge systems is discussed. The relation between the measured (apparent) substrate affinity constant and diffusion processes is outlined.

Biofilms↗
PubMed2006

Long-term persistence of a nutrient-starved biofilm in a limestone fracture.

The persistence of biofilms is a key element of the biobarrier concept applied to fractured rock. After a 43-day biostimulation with molasses for the biofilm growth (phase I), the effects of a 179-day starvation on the persistence of the biofilm (phase II) were investigated in a single-fractured limestone apparatus equipped to detect small changes in hydraulic conductivity (K) (cm min(-1)). The K in the central fracture section (Kf) decreased by 4.6 logs between days 0 and 167 and increased by 1.3 logs between days 167 and 222, leading to an overall 3.3-logs reduction for the 222-day experiment. Accumulation of rod- and filamentous-shaped bacteria and deposition of minerals were thought to account for the decrease in Kf. Even though the filamentous bacterial cells possibly enhanced the stability of the developing biofilm, increases in Kf became more frequent after 100 days of operation. This decrease in stability was presumably related to sloughing events, which were in turn attributed to a combination of processes, including nutrient deprivation, the release of deposited minerals, decreasing fluid flowrate, and endogenous decay as part of biofilm ageing. This study indicates that biofilms developed in fractures can persist for extended periods at reduced K when exposed to a long-term starvation.

Bacteria↗
PubMed2006

Bacteria removal in septic effluent: influence of biofilm and protozoa.

Numerous biological, physical and chemical parameters are involved in the retention and removal of bacteria in wastewater treatment systems. Biological parameters, such as biofilms and protozoa grazing activity, are often mentioned but few studies provide a better understanding of their influence. In this study, the effect of bacterivorous protozoa on pathogenic indicator bacteria removal was investigated in septic effluent and in the presence of a biofilm coating glass slides. Endogenous bacteria from septic effluent were quantified. First, bacteria removal was compared between septic effluents treated or not with an inhibitor of protozoa (cycloheximide). The mortality rates were 10 times lower in treated effluent (96 CFU mL(-1) d(-1)) than in untreated effluent (1100 CFU mL(-1) d(-1)). Secondly, the efficiency of bacteria removal was studied (i) with a biofilm surface and active protozoa, (ii) with a biofilm surface and inactivated protozoa, (iii) with a clean surface. Protozoa in the presence of a biofilm were responsible for 60% of bacteria removal. Biofilm without protozoa and a clean surface each removed similar quantities of bacteria. Grazing by protozoa could be an important biological mechanism for bacterial elimination in wastewater treatment systems.

Animals↗
PubMed2026

Building biofilms for saline hydrogenotrophic denitrification from contrasting origins: Convergent acclimation, divergent performance.

Hydrogenotrophic denitrification is promising for deep nitrogen removal from saline, low-C/N wastewaters, but rapid establishment of stable biofilms at high salinity remains challenging. Here, two saline-adapted inocula from two representative, functionally contrasting habitats-a functionally-diversified inoculum from mangrove sediment and a functionally-focused inoculum from seabed sediment-were acclimated in parallel H2-based membrane biofilm reactors at constant 3.5% salinity. The Diverse-derived biofilm required 80 d to reach steady state and achieved only partial denitrification with 61.1% nitrate removal and considerable nitrite accumulation. In contrast, the Focus-derived biofilm rapidly established complete denitrification within ∼40 d, which was maintained for >50 d, with effluent NOx- below 1 mg-N·L-1 and 98.7% nitrate removal. Microbiome analyses showed that identical operation promoted convergence in community structure and enriched similar community-level functional potentials. However, genome-resolved analysis revealed distinct source-dependent functional organization among dominant microbial populations. Complete denitrifiers co-encoding denitrifying, hydrogenotrophic, and autotrophic functions were preferentially enriched in the Focus-derived biofilm, whereas these functions remained partitioned among different dominant populations in the Diverse-derived biofilm, coinciding with less complete denitrification. These findings indicate that saline hydrogenotrophic denitrification performance depends not only on which functions are enriched at the community level, but also on how key functions become organized among microbial populations, providing a previously overlooked criterion for inoculum selection in saline biological nitrogen control.

Complete denitrification↗
PubMed2000

Electrical enhancement of Streptococcus gordonii biofilm killing by gentamicin.

This electrical enhancement was demonstrated in an in vitro model. Streptococcus gordonii biofilms were grown for 6 days in continuous-flow reactors on one-tenth strength trypticase peptone broth. The biofilms attained a mean areal cell density of 2.4 x 10(8) c.f.u./cm2 and a thickness of approx. 19 microm. Biofilms exhibited characteristic resistance to killing by an antibiotic. When treated with 2 microg/ml gentamicin for 24 h, they exhibited a 0.84 log reduction in viable cell numbers; a 4.7 log reduction was measured in a planktonic culture. Killing of planktonic bacteria by this treatment was reduced to 1.2 log when an oxygen-scavenging enzyme was added to the medium. When a 2-mA direct current was applied during antibiotic treatment, biofilm killing increased to a 4.3 log reduction. Electrical current alone caused a 1.9 log reduction in biofilm cell counts. It is suggested that gentamicin was less effective against Strep. gordonii under anaerobic conditions than it was under aerobic conditions and that this can explain both the reduced susceptibility of the biofilm (due to oxygen depletion) and electrical enhancement of efficacy (due to oxygen generation by electrolysis).

Aerobiosis↗
PubMed2001

Removal of easily biodegradable organic compounds by drinking water biofilms: analysis of kinetics and mass transfer.

This paper evaluates the rate of utilization of easily biodegradable organic compounds by drinking water biofilms. Tap water, which had been filtered through biologically active granular activated carbon, was used as an innoculum for biofilm growth in annular reactors (ARs). Synthetic cocktails of easily biodegradable material in the concentration range of 50-2,000 mgC/m3 were used as substrate for biofilm growth. Influent and effluent aggregate concentrations of biodegradable organic matter (BOM) were calculated by adding the measurable BOM components on a mass carbon basis. The aggregate BOM values were used for calculating the observed Damköhler number and Theile modulus (based on a reaction rate per unit surface area), which were used to determine whether external or internal mass transfer limited BOM removal. For all of the experimental trials, it was shown that neither external nor internal mass transfer limited BOM removal. Because the biofilms in this research are thin and the fact that mass transfer is not limiting, it was assumed that the bulk BOM concentration was approximately equal to the average BOM concentration in the biofilm. A linear model was obtained for the aggregate BOM flux and the product of the effluent BOM concentration and the biofilm density. The slope or the areal biodegradation rate (ka) for the aggregate BOM was 0.033 m/h, as determined through a linear regression.

Biodegradation, Environmental↗
PubMed2001

Selective degradation of ibuprofen and clofibric acid in two model river biofilm systems.

A field survey indicated that the Elbe and Saale Rivers were contaminated with both clofibric acid and ibuprofen. In Elbe River water we could detect the metabolite hydroxy-ibuprofen. Analyses of the city of Saskatoon sewage effluent discharged to the South Saskatchewan river detected clofibric acid but neither ibuprofen nor any metabolite. Laboratory studies indicated that the pharmaceutical ibuprofen was readily degraded in a river biofilm reactor. Two metabolites were detected and identified as hydroxy- and carboxy-ibuprofen. Both metabolites were observed to degrade in the biofilm reactors. However, in human metabolism the metabolite carboxy-ibuprofen appears and degrades second whereas the opposite occurs in biofilm systems. In biofilms the pharmacologically inactive stereoisomere of ibuprofen is degraded predominantly. In contrast, clofibric acid was not biologically degraded during the experimental period of 21 days. Similar results were obtained using biofilms developed using waters from either the South Saskatchewan or Elbe River. In a sterile reactor no losses of ibuprofen were observed. These results suggested that abiotic losses and adsorption played only a minimal role in the fate of the pharmaceuticals in the river biofilm reactors.

Biodegradation, Environmental↗
PubMed2002

Adsorption of p-chlorophenol by biofilm components.

Through batch equilibrium experiments under the conditions of temperature 25 degrees C, pH values of 2.7, 5.3 and 6.1, the p-chlorophenol (4-cp) adsorption to biofilm components was investigated in this study. The contributions of biofilm components to 4-cp adsorption were discussed by comparing four adsorption systems, i.e. 4-cp adsorbed by model suspended particulate matter (kaolin) with biofilm coating, bacteria, bacterial exopolysaccharide (EPS) and kaolin, respectively. Langmuir and Freundlich isotherm equations were used to evaluate the experiment data. All the four adsorptions fitted for the two equations. Equilibrium isotherms were obtained for 4-cp adsorption on different adsorbents. The kinetic characteristics of 4-cp adsorption by biofilm components and the effect of pH on the kinetic process were investigated. The time to reach the highest adsorption amount and near equilibrium state in the four systems was different. The 4-cp adsorption by kaolin with biofilm coating reached near equilibrium at 60 min at pH 6.1. The 4-cp adsorption by EPS and kaolin reached near equilibrium at 150 and 180 min, respectively. But the 4-cp adsorption by bacteria showed no evident near equilibrium during 3 h in the experiment. The impact of pH value on the adsorption was also examined. The adsorption amount slightly increased with increasing pH from 2.7 to 6.1 for the adsorption systems of bacterial EPS and kaolin, but it slightly decreased in the systems of kaolin with biofilm coating and bacteria.

Adsorption↗
PubMed2003

COD and nitrogen removal by biofilms growing on gas permeable membranes.

A bioreactor was constructed and used to treat a synthetic wastewater containing ammonium acetate and trace nutrients for about 190 days. The reactor was aerated by means of bundles of gas-permeable hollow-fiber membranes that were installed in the reactor. The membranes provided a specific surface area of 422 m(2)/m(3) and the external surface of the membranes rapidly became covered in an active biofilm. The membrane bundles were agitated by an internal gas recycle. The gas bubbles in the water encouraged fiber-fiber contact and were intended to control biofilm growth. Chemical oxygen demand (COD) removals in excess of 95% were achieved in a 6h nominal detention time. Nitrification developed rapidly and complete oxidation of the influent ammonium was evident within 20 days. Even though the reactor was equipped with a large membrane surface area, the oxygen was consumed within the biofilm growing on the membrane surface. As a result, the external dissolved oxygen (DO) dropped to zero and the reactor was able to support essentially complete denitrification. After about 3 months of operation the reactor showed excellent removals of both COD and inorganic nitrogen but the performance could not be sustained. Excess biofilm accumulation eventually contributed to a deterioration in process performance. This study demonstrates that while membrane aeration can provide simultaneous BOD and N removal in the same reactor, the membrane modules/bioreactor must be designed to allow for the development of thick biofilms. In addition, options for controlling the biofilm thickness need to be investigated.

Biofilms↗
PubMed2001

Phenotype characterization of genetically defined microorganisms and growth of bacteriophage in biofilms.

Phenotypic characterization will be a pivotal aspect of future research in understanding the biofilm mode of growth. We hope that the concepts and techniques presented in this chapter will benefit other investigators in this field. Although initial studies will necessarily involve monocultures, eventually mixed culture work will have to be performed to understand biofilm growth in the natural environment. As the study of biofilm-phage interactions is new, there is considerable fundamental work that needs to be addressed. Here, we anticipate that some phage are better adapted to growth in biofilms, some are adept in growing in mixed culture biofilms, and others are better adapted to infecting planktonic organisms. Whereas biofilms are now widely accepted as a fundamental aspect of microbial growth in nature, the field of phage ecology is quite new and an exciting challenge for the future.

Bacteria↗
PubMed1995

Synergy between protamine and vancomycin in the treatment of Staphylococcus epidermidis biofilms.

OBJECTIVES: To test for synergy between protamine and vancomycin by analyzing their bactericidal activities against slime-producing Staphylococcus epidermidis ATCC 35983 under planktonic and biofilm conditions. METHODS: We evaluated the activity of vancomycin and protamine separately against planktonic S epidermidis in broth by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for each agent according to the standard macrobroth dilution method. To assess the possibility of synergy between these two agents, planktonic S epidermidis was exposed to vancomycin and protamine together in varying concentrations. Biofilms containing S epidermidis were then prepared and subjected to incubation with vancomycin and protamine separately as well as combined in varying concentrations. The bacterial viability of S epidermidis in the planktonic and biofilm phases after exposure to these two agents was assessed by qualitative culture and determination of viable colony blots. RESULTS: Standard antibacterial susceptibility tests revealed that the MICs of protamine and vancomycin were 1 and 2 micrograms/mL, respectively, and their MBCs were 4 micrograms/mL. The MICs were unchanged when protamine and vancomycin were combined in varying concentrations. Neither agent exhibited significant bactericidal activity against S epidermidis in the biofilm phase at concentrations < or = 32 micrograms/mL. However, a combination of both agents, each at 32 micrograms/mL, resulted in a 7-log decrease in viable bacterial counts. CONCLUSIONS: Protamine alone exhibited significant antibacterial activity against planktonic S epidermidis. No synergy was noted between protamine and vancomycin against S epidermidis in the planktonic phase. However, synergy was demonstrated when a combination of protamine and vancomycin was used on S epidermidis in the biofilm phase. Thus, protamine shows promise as an adjunctive agent to vancomycin in the treatment of S epidermidis in biofilms.

Biofilms↗
PubMed2003

The dependence of quorum sensing on the depth of a growing biofilm.

In a process called quorum sensing, bacteria monitor their population density via extracellular signaling molecules and modulate gene expression accordingly. In this paper, a one-dimensional model of a growing Pseudomonas aeruginosa biofilm is examined. Quorum sensing has been included in the model through equations describing the production, degradation, and diffusion of the signaling molecules, acyl-homoserine lactones, in the biofilm. From this model, we are able to make some important observations about quorum sensing. First, in order for quorum sensing to initiate near the substratum, in accordance with experimental observations, the model suggests that cells in oxygen-deficient regions of the biofilm must still be synthesizing the signal compound. Second, the induction of quorum sensing is related to a critical biofilm depth; once the biofilm grows to the critical depth, quorum sensing is induced. Third, the critical biofilm depth varies with the pH of the surrounding fluid. Of particular interest is the prediction of a critical pH threshold, above which quorum sensing is not possible at any depth. These results highlight the importance of careful study of the relationship among metabolic activity of the bacterium, signal synthesis, and the chemistry of the surrounding environment.

Biofilms↗