Metagenomic and metaproteomic analyses of Accumulibacter phosphatis‐enriched floccular and granular biofilm

Biofilms are ubiquitous in nature, forming diverse adherent microbial communities that perform a plethora of functions. Here we operated two laboratory‐scale sequencing batch reactors enriched with Candidatus Accumulibacter phosphatis (Accumulibacter) performing enhanced biological phosphorus removal. Reactors formed two distinct biofilms, one floccular biofilm, consisting of small, loose, microbial aggregates, and one granular biofilm, forming larger, dense, spherical aggregates. Using metagenomic and metaproteomic methods, we investigated the proteomic differences between these two biofilm communities, identifying a total of 2022 unique proteins. To understand biofilm differences, we compared protein abundances that were statistically enriched in both biofilm states. Floccular biofilms were enriched with pathogenic secretion systems suggesting a highly competitive microbial community. Comparatively, granular biofilms revealed a high‐stress environment with evidence of nutrient starvation, phage predation pressure, and increased extracellular polymeric substance and cell lysis. Granular biofilms were enriched in outer membrane transport proteins to scavenge the extracellular milieu for amino acids and other metabolites, likely released through cell lysis, to supplement metabolic pathways. This study provides the first detailed proteomic comparison between Accumulibacter‐enriched floccular and granular biofilm communities, proposes a conceptual model for the granule biofilm, and offers novel insights into granule biofilm formation and stability.     

Testing the Efficiency of Biofilms Formed on Natural Substratum,Coyonoxtle (Opuntia Imbricata), for the Anaerobic Degradation of Aromatic Compounds

In this study, previously developed anaerobic microbial consortia capable of degrading aromatic compounds were used to develop biofilms on a natural material, coyonoxtle (Opuntia imbricata), which is abundantly available in North Mexico. The developed biofilms were evaluated for their efficiency in the biodegradation of different aromatic compounds, viz., phenol, catechol, 4‐aminobenzoic acid and p‐phenylenediamine in batch reactors. It was observed that in reactors with biofilms a more than 90 % COD removal and a concomitant production of methane could be obtained. But the rate of COD removal and methane production varied depending upon the type of biofilm used. Rumen‐derived biofilms demonstrated a lag phase of 7 to 14 days, whereas sludge‐derived biofilms exhibited a lag phase of more than three weeks. Between the biofilms from two sources, rumen‐derived biofilms showed a higher COD removal and methane production than sludge‐derived biofilms. When biofilm reactors were compared with reactors containing freely suspended consortia, it was evident that both rumen– and sludge‐derived biofilm reactors exhibited a two‐fold higher COD removal and methane production. Based on the results obtained, it can be concluded that coyonoxtle has the potential for use as a substratum.     

Redox-stratification controlled biofilm (ReSCoBi) for completely autotrophic nitrogen removal: the effect of co- versus counter-diffusion on reactor performance

A multi-population biofilm model for completely autotrophic nitrogen removal was developed and implemented in the simulation program AQUASIM to corroborate the concept of a redox-stratification controlled biofilm (ReSCoBi). The model considers both counter- and co-diffusion biofilm geometries. In the counter-diffusion biofilm, oxygen is supplied through a gas-permeable membrane that supports the biofilm while ammonia (NH(4)(+)) is supplied from the bulk liquid. On the contrary, in the co-diffusion biofilm, both oxygen and NH(4)(+) are supplied from the bulk liquid. Results of the model revealed a clear stratification of microbial activities in both of the biofilms, the resulting chemical profiles, and the obvious effect of the relative surface loadings of oxygen and NH(4)(+) (J(O(2))/J(NH(4)(+))) on the reactor performances. Steady-state biofilm thickness had a significant but different effect on T-N removal for co- and counter-diffusion biofilms: the removal efficiency in the counter-diffusion biofilm geometry was superior to that in the co-diffusion counterpart, within the range of 450-1,400 microm; however, the efficiency deteriorated with a further increase in biofilm thickness, probably because of diffusion limitation of NH(4)(+). Under conditions of oxygen excess (J(O(2))/J(NH(4)(+)) > 3.98), almost all NH(4)(+) was consumed by aerobic ammonia oxidation in the co-diffusion biofilm, leading to poor performance, while in the counter-diffusion biofilm, T-N removal efficiency was maintained because of the physical location of anaerobic ammonium oxidizers near the bulk liquid. These results clearly reveal that counter-diffusion biofilms have a wider application range for autotrophic T-N removal than co-diffusion biofilms.     

Next-Generation Pyrosequencing Analysis of Microbial Biofilm Communities on Granular Activated Carbon in Treatment of Oil Sands Process-Affected Water

The development of biodegradation treatment processes for oil sands process-affected water (OSPW) has been progressing in recent years with the promising potential of biofilm reactors. Previously, the granular activated carbon (GAC) biofilm process was successfully employed for treatment of a large variety of recalcitrant organic compounds in domestic and industrial wastewaters. In this study, GAC biofilm microbial development and degradation efficiency were investigated for OSPW treatment by monitoring the biofilm growth on the GAC surface in raw and ozonated OSPW in batch bioreactors. The GAC biofilm community was characterized using a next-generation 16S rRNA gene pyrosequencing technique that revealed that the phylum Proteobacteria was dominant in both OSPW and biofilms, with further in-depth analysis showing higher abundances of Alpha- and Gammaproteobacteria sequences. Interestingly, many known polyaromatic hydrocarbon degraders, namely, Burkholderiales, Pseudomonadales, Bdellovibrionales, and Sphingomonadales, were observed in the GAC biofilm. Ozonation decreased the microbial diversity in planktonic OSPW but increased the microbial diversity in the GAC biofilms. Quantitative real-time PCR revealed similar bacterial gene copy numbers (>10⁹ gene copies/g of GAC) for both raw and ozonated OSPW GAC biofilms. The observed rates of removal of naphthenic acids (NAs) over the 2-day experiments for the GAC biofilm treatments of raw and ozonated OSPW were 31% and 66%, respectively. Overall, a relatively low ozone dose (30 mg of O₃/liter utilized) combined with GAC biofilm treatment significantly increased NA removal rates. The treatment of OSPW in bioreactors using GAC biofilms is a promising technology for the reduction of recalcitrant OSPW organic compounds.     

Persister cells, the biofilm matrix and tolerance to metal cations in biofilm and planktonic Pseudomonas aeruginosa

In this study, we examined Pseudomonas aeruginosa ATCC 27853 biofilm and planktonic cell susceptibility to metal cations. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) required to eradicate 100% of the planktonic population (MBC 100), and the minimum biofilm eradication concentration (MBEC) were determined using the MBEC trade mark-high throughput assay. Six metals - Co(2+), Ni(2+), Cu(2+), Zn(2+), Al(3+) and Pb(2+)- were each tested at 2, 4, 6, 8, 10 and 27 h of exposure to biofilm and planktonic cultures grown in rich or minimal media. With 2 or 4 h of exposure, biofilms were approximately 2-25 times more tolerant to killing by metal cations than the corresponding planktonic cultures. However, by 27 h of exposure, biofilm and planktonic bacteria were eradicated at approximately the same concentration in every instance. Viable cell counts evaluated at 2 and 27 h of exposure revealed that at high concentrations, most of the metals assayed had killed greater than 99.9% of biofilm and planktonic cell populations. The surviving cells were propogated in vitro and gave rise to biofilm and planktonic cultures with normal sensitivity to metals. Further, retention of copper by the biofilm matrix was investigated using the chelator sodium diethlydithiocarbamate. Formation of visible brown metal-chelates in biofilms treated with Cu(2+) suggests that the biofilm matrix may coordinate and sequester metal cations from the aqueous surroundings. Overall, our data suggest that both metal sequestration in the biofilm matrix and the presence of a small population of 'persister' cells may be contributing factors in the time-dependent tolerance of both planktonic cells and biofilms to high concentrations of metal cations.     

Heavy metal tolerance in marine strains of Yarrowia lipolytica

Heavy metal tolerance of two marine strains of Yarrowia lipolytica was tested on solid yeast extract peptone dextrose agar plates. Based on minimum inhibitory concentration esteems, it is inferred that the two strains of Y. lipolytica were tolerant to heavy metals such as Pb(II), Cr(III), Zn(II), Cu(II), As(V), and Ni(II) ions. The impact of various heavy metal concentrations on the growth kinetics of Y. lipolytica was likewise assessed. With increased heavy metal concentration, the specific growth rate was reduced with delayed doubling time. Furthermore, biofilm development of both yeasts on the glass surfaces and in microtitre plates was assessed in presence of different heavy metals. In microtitre plates, a short lag phase of biofilm formation was noticed without the addition of heavy metals in yeast nitrogen base liquid media. A lag phase was extended over increasing metal concentrations of media. Heavy metals like Cr(VI), Cd(II), and As(V) are contrastingly influenced on biofilms’ formation of microtitre plates. Other heavy metals did not much influence on biofilms development. Thus, biofilm formation is a strategy of Y. lipolytica under stress of heavy metals has significance in bioremediation process for recovery of heavy metals from contaminated environment.

     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l(-1) caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

Sodium dodecyl sulfate allows the persistence and recovery of biofilms of Pseudomonas fluorescens formed under different hydrodynamic conditions

The effect of the anionic surfactant sodium dodecyl sulfate (SDS) on Pseudomonas fluorescens biofilms was investigated using flow cell reactors with stainless steel substrata, under turbulent (Re = 5200) and laminar (Re = 2000) flow. Steady-state biofilms were exposed to SDS in single doses (0.5, 1, 3 and 7 mM) and biofilm respiratory activity and mass measured at 0, 3, 7 and 12 h after the SDS application. The effect of SDS on biofilm mechanical stability was assessed using a rotating bioreactor. Whilst high concentrations (7 mM) of SDS promoted significant biofilm inactivation, it did not significantly reduce biofouling. Turbulent and laminar flow-generated biofilms had comparable susceptibility to SDS application. Following SDS exposure, biofilms rapidly recovered over the following 12 h, achieving higher respiratory activity values than before treatment. This phenomenon of post-treatment recovery was more pronounced for turbulent flow-generated biofilms, with an increase in SDS concentration. The mechanical stability of the biofilms increased with surfactant application, except for SDS concentrations near the critical micellar concentration, as measured by biofilm removal due to an increase in external shear stress forces. The data suggest that although SDS exerts antimicrobial action against P. fluorescens biofilms, even if only partial and reversible, it had only limited antifouling efficacy, increasing biofilm mechanical stability at low concentrations and allowing significant and rapid recovery of turbulent flow-generated biofilms.     

Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 microm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 microm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 microm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

Metal immobilisation by biofilms: Mechanisms and analytical tools

In biofilm environments, heavy metal and radionuclide pollutants are removed by a variety of mechanisms, including biosorption, precipitation as sulfides or phosphates and microbial reductive precipitation. Even if the elemental composition and localization of the precipitate trapped in the biofilm is well described thanks to spectroscopic and microscopic techniques, this review highlights that little is known about metal immobilisation mechanisms in microbial biofilms, i.e., mass transfer of metals, mechanisms involved in (bio)sorption and precipitation and the influence of physicochemical micro-environments within the biofilm matrix. The review shows the advantage of using a combination of different techniques to evaluate the fate of metals within microbial biofilms. By combining a variety of techniques (e.g., selective extraction, microscopy, spectroscopy and miniaturised sensors ...), it is possible to gain high-resolution structural and chemical information of biofilms on a level of the individual cell. This approach will facilitate the characterization of the metal immobilisation sites and the metal sorption and (bio)crystallisation mechanisms in biofilms. The results provided by the combination of these techniques will allow to predict the amount of metal accumulation in biofilms as well as their chemical speciation. This review demonstrates that an interdisciplinary approach is required to study metal fate within the biofilm matrix. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhancement of Anaerobic Digestion Using Particulate Growth Systems

Attached media reactors are used for enhancement of wastewater treatment processes including anaerobic condition. Selection of a suitable biofilm carrier is a compelling method to improve anaerobic digestion systems. This study investigates the performance of four fibrous biofilms installed in batch biogas reactors for treatment of cow manure. BioCords HS1, HS2, LS1, and LS2 are manufactured by Bishop Water Technologies, ON, Canada. Effluents and attached growth media were analyzed after batch experiment; methane production, methane yield, transfer efficiencies, organic and solid removal efficiencies, pH, and attached volatile suspended solid (VSS) were measured; VSS attached to biofilms mainly correlated with the specific surface area of each biofilm. Additionally, SEM (scanning electron microscopy) was used for further understanding of biofilm formation process for BioCords and the dissimilarity in their performance. The results indicated that BioCord LS2 had positive impact on achieving higher methane production and removal efficiencies compared to other support media utilized in batch reactors. It was also demonstrated from the experiment that BioCord LS2 potentially could generate higher methane production than conventional batch bioreactor.     

A repeatable laboratory method for testing the efficacy of biocides against toilet bowl biofilms

AIMS: The purpose of this study was to develop a laboratory biofilm growth reactor system that simulated the toilet bowl environment and which could be used for biocide efficacy testing. METHODS AND RESULTS: A microbial biofilm reactor system incorporating intermittent flow and nutrient provision was designed. The reactor system was open to the air and was inoculated with organisms collected from toilet bowl biofilms. Once per hour, reactors were supplied with a nutrient solution for a period of 5 min, then flushed and refilled with tap water or tap water amended with chlorine. Quantitative measures of the rate and extent of biofilm accumulation were defined. Biofilm accumulated in untreated reactors to cell densities of 108 cfu cm-2 after approximately 1 week. Biofilm accumulation was also observed in reactors in the continuous presence of several milligrams per litre of free chlorine. Repeatability standard deviations for the selected efficacy measures were low, indicating high repeatability between experiments. Log reduction values of viable cell numbers were within ranges observed with standard suspension and hard surface disinfection tests. Biofilm accumulated in laboratory reactors approximately seven times faster than it did in actual toilet bowls. The same ranking was achieved in tests between laboratory biofilms and field-grown biofilms with three of the four measures, using three different concentrations of chlorine. CONCLUSION: This reactor system has been shown to simulate, in a repeatable way, the accumulation of bacterial biofilm that occurs in toilet bowls. The results demonstrate that this system can provide repeatable assays of the efficacy of chlorine against those biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The laboratory biofilm reactor system described herein can be used to evaluate potential antimicrobial and antifouling treatments for control of biofilm formation in toilet bowls.     

Determination of Spatial Distributions of Zinc and Active Biomass in Microbial Biofilms by Two-Photon Laser Scanning Microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 μm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 μm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 μm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

Metal resistance in Candida biofilms

Yeasts are often successful in metal-polluted environments; therefore, the ability of biofilm and planktonic cell Candida tropicalis to endure metal toxicity was investigated. Fifteen water-soluble metal ions, chosen to represent groups 6A to 6B of the periodic table, were tested against this organism. With in vitro exposures as long as 24 h, biofilms were up to 65 times more tolerant to killing by metals than corresponding planktonic cultures. Of the most toxic heavy metals tested, only very high concentrations of Hg2+, CrO4 (2-) or Cu2+ killed surface-adherent Candida. Metal-chelator precipitates could be formed in biofilms following exposure to the heavy metals Cu2+ and Ni2+. This suggests that Candida biofilms may adsorb metal cations from their surroundings and that sequestration in the extracellular matrix may contribute to resistance. We concluded that biofilm formation may be a strategy for metal resistance and/or tolerance in yeasts.     

Performance and microbial analysis of defined and non-defined inocula for the removal of dimethyl sulfide in a biotrickling filter

The performance and microbial communities of three differently inoculated biotrickling filters removing dimethyl sulfide (DMS) were compared. The biotrickling filters were inoculated with Thiobacillus thioparus TK-m (THIO), sludge (HANDS) and sludge + T. thioparus TK-m + Hyphomicrobium VS (HANDS++), respectively. The criteria investigated were length of the start-up period, the maximum elimination capacity, and the effects of intermittent loading rates, low pH, peak loading and very low loading rate on the DMS removal efficiency. The HANDS++ reactor exhibited the best performance considering all treatments. HANDS performed almost equally well as HANDS++, except during the determination of the EC(max), while THIO was generally the least efficient. During stable DMS loading at concentrations of 20 ppmv or lower, all reactors exhibited similar and high removal efficiencies (>99%). Denaturing gradient gel electrophoresis (DGGE) analysis showed the establishment of T. thioparus in the biofilm of all reactors, but not of Hyphomicrobium VS. Quantitative monitoring of the introduced bacterial strains was performed with a newly developed real-time PCR protocol. Initially, the inoculated strains were exclusively found in the reactors in which they were added. Afterwards, however, both strains developed in the biofilm of all three reactors, although T. thioparus attained higher cell densities than Hyphomicrobium. The presence of T. thioparus in THIO was related with the DMS loading rates that were applied, in the sense that intermittent DMS loading and very low DMS loading rates (0.5 ppmv) induced a decrease in gene copy numbers. Real-time PCR and DGGE both gave consistent results regarding the presence of Hyphomicrobium VS and Thiobacillus thioparus TK-m in the reactors. Only real-time PCR could be used to detect bacteria comprising of less than 1.4% of the total bacterial community ( approximately 10(5) copies ring(-1)).     

Effects of carbon and oxygen limitations and calcium concentrations on biofilm removal processes

Bacterial biofilm removal processes due to shear and catastrophic sloughing have been investigated in a turbulent flow system under conditions of carbon versus oxygen substrate limitations and varying aqueous phase calcium concentrations. Biofilm cellular and extracellular polymer carbon, total biofilm carbon and mass, and biofilm calcium concentrations are measured for pure culture biofilms of the facultative aerobe, Pseudomonas putida ATCC 11172. Results indicate oxygen-limited biofilms reach a higher steady-state biofilm organic carbon level than carbon-limited biofilms. Oxygen-limited biofilms also exhibit (1) a higher extracellular polymer-carbon: cell-carbon ratio throughout biofilm development and (2) a higher biofilm calcium content than carbon-limited biofilms. Increasing aqueous phase calcium concentrations increase the amount of biofilm calcium in both cases; the rate of calcium accumulation in oxygen-limited biofilms increases with increasing liquid phase calcium concentrations over the entire range studied while the rates of calcium accumulation in carbon-limited biofilms appear independent of aqueous phase calcium concentrations above 11.0 mg/L. Oxygen-limited biofilms with their higher extracellular polymer and calcium content exhibit shear removal rates that are 20-40% of those observed for carbon-limited biofilms. However, it is the oxygen-limited biofilms that experience catastrophic sloughing events. The carbon-limited biofilms studied here never sloughed even if subjected to intentional long-term deprivation of all nutrients. Reduced shear removal and the susceptibility to sloughing of the oxygen-limited biofilms are attributed to their more cohesive structure bought about by their relatively greater extracellular polymer production.     

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.     

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.     

Heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa

A study was undertaken to examine the effects of the heavy metals copper, lead, and zinc on biofilm and planktonic Pseudomonas aeruginosa. A rotating-disk biofilm reactor was used to generate biofilm and free-swimming cultures to test their relative levels of resistance to heavy metals. It was determined that biofilms were anywhere from 2 to 600 times more resistant to heavy metal stress than free-swimming cells. When planktonic cells at different stages of growth were examined, it was found that logarithmically growing cells were more resistant to copper and lead stress than stationary-phase cells. However, biofilms were observed to be more resistant to heavy metals than either stationary-phase or logarithmically growing planktonic cells. Microscopy was used to evaluate the effect of copper stress on a mature P. aeruginosa biofilm. The exterior of the biofilm was preferentially killed after exposure to elevated concentrations of copper, and the majority of living cells were near the substratum. A potential explanation for this is that the extracellular polymeric substances that encase a biofilm may be responsible for protecting cells from heavy metal stress by binding the heavy metals and retarding their diffusion within the biofilm.     

Method for Studying Microbial Biofilms in Flowing-Water Systems

A method for the study of microbial biofilms in flowing-water systems was developed with special reference to the flow conditions in electrochemical concentration cells. Seawater was circulated in a semiclosed flow system through biofilm reactors (3 cm s⁻¹) with microscope cover slips arranged in lamellar piles parallel with the flow. At fixed time intervals cover slips with their biofilm were removed from the pile, stained with crystal violet, and mounted on microscope slides. The absorbances of the slides were measured at 590 nm and plotted against time to give microbial biofilm development. From calibration experiments a staining time of 1 min and a rinse time of 10 min in a tap water flow (3 cm s⁻¹) were considered sufficient. When an analysis of variance was performed on biofilm development data, 78% of the total variance was found to be due to random natural effects; the rest could be explained by experimental effects. The absorbance values correlated well with protein N, dry weight, and organic weight in two biofilm experiments, one with a biofilm with a high (75%) and one with a low (~25%, normal) inorganic content. Comparisons of regression lines revealed that the absorbance of the stained biofilms was an estimate closely related to biofilm dry weight.