Investigation of natural biofilms formed during the production of drinking water from surface water embankment filtration

Populations of bacteria in biofilms from different sites of a drinking water production system were analysed. Polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analyses revealed changing DNA band patterns, suggesting a population shift during bank filtration and processing at the waterworks. In addition, common DNA bands that were attributed to ubiquitous bacteria were found. Biofilms even developed directly after UV disinfection (1-2m distance). Their DNA band patterns only partly agreed with those of the biofilms from the downstream distribution system. Opportunistic pathogenic bacteria in biofilms were analysed using PCR and Southern blot hybridisation (SBH). Surface water appeared to have a direct influence on the composition of biofilms in the drinking water distribution system. In spite of preceding filtration and UV disinfection, opportunistic pathogens such as atypical mycobacteria and Legionella spp. were found in biofilms of drinking water, and Pseudomonas aeruginosa was detected sporadically. Enterococci were not found in any biofilm. Bacterial cell counts in the biofilms from surface water to drinking water dropped significantly, and esterase and alanine-aminopeptidase activity decreased. beta-glucosidase activity was not found in the biofilms. Contrary to the results for planktonic bacteria, inhibitory effects were not observed in biofilms. This suggested an increased tolerance of biofilm bacteria against toxic compounds.     

Application of a molecular biology concept for the detection of DNA damage and repair during UV disinfection

As nucleic acids are major targets in bacteria during standardised UV disinfection (254 nm), inactivation rates also depend on bacterial DNA repair. Due to UV-related DNA modifications, PCR-based approaches allow for a direct detection of DNA damage and repair during UV disinfection. By applying different primer sets, the correlation between amplicon length and PCR amplification became obvious. The longer the targeted DNA fragment was, the more UV-induced DNA lesions inhibited the PCR. Regeneration of Pseudomonas aeruginosa, Enterococcus faecium, and complex wastewater communities was recorded over a time period of 66 h. While phases of intensive repair and proliferation were found for P. aeruginosa, no DNA repair was detected by qPCR in E. faecium. Cultivation experiments verified these results. Despite high UV mediated inactivation rates original wastewater bacteria seem to express an enhanced robustness against irradiation. Regeneration of dominant and proliferation of low-abundant, probably UV-resistant species contributed to a strong post-irradiation recovery accompanied by a selection for β-Proteobacteria.     

A field study evaluation for mitigating biofouling with chlorine dioxide or chlorine integrated with UV disinfection

The drinking water industry is continually seeking innovative disinfection strategies to control biofouling in transmission systems. This research, conducted in collaboration with the East Bay Municipal Utility District (EBMUD) in California, compared the efficacy of chlorine dioxide (ClO2) to free chlorine (Cl2) with and without pre-treatment with low-pressure ultraviolet (UV) light for biofilm control. An additional goal was to determine disinfection by-product (DBP) formation with each disinfection strategy. Annular reactors (ARs) containing polycarbonate coupons were used to simulate EBMUD's 90-mile aqueduct that transports surface water from a source reservoir to treatment facilities. ARs were dosed with chemical disinfectants to achieve a residual of 0.2 mg/L, which is a typical value mid-way in the aqueduct. The experiment matrix included four strategies of disinfection including UV/ClO2, ClO2, UV/Cl2 and Cl2. Two ARs acted as controls and received raw water (RW) or UV-treated water. The data presented show that the UV/ClO2 combination was most effective against suspended and attached heterotrophic (heterotrophic plate count, HPC) bacteria with 3.93 log and 2.05 log reductions, respectively. ClO2 was more effective than Cl2 at removing suspended HPC bacteria and similarly effective in biofilm bacterial removal. UV light alone was not effective in controlling suspended or biofilm bacteria compared to treatment with ClO2 or Cl2. Pre-treatment with UV was more effective overall for removal of HPC bacteria than treating with corresponding chemical disinfectants only; however, it did not lower required chemical dosages. Therefore, no significant differences were observed in DBP concentrations between ARs pre-treated with UV light and ARs not pre-treated. Disinfection with ClO2 produced fewer total trihalomethanes (TTHMs) and haloacetic acids (HAAs) than chlorination but did produce low levels of chlorite. These data indicate that replacing Cl2 with ClO2 would further control microbiological re-growth and minimize TTHM and HAA formation, but may introduce other DBPs.     

Interactions of nanosilver with Escherichia coli cells in planktonic and biofilm cultures

Biofilms are often more resistant to toxic chemicals such as heavy metals and antimicrobial agents than planktonic cells. Nanosilver has a broad range of applications with strong antimicrobial activity. However, biofilm susceptibility to nanosilver toxicity is not well understood. We studied the bacterial activity in planktonic or biofilm cultures after nanosilver exposure using oxygen quenching fluorescence-based microrespirometry. We also determined the aggregation behavior and the spatial distribution of nanosilver having red fluorescence in biofilms of Escherichia coli expressing green fluorescent protein. At the same bacterial concentrations (3 × 10⁸ CFU/mL), biofilms were about four times more resistant to nanosilver inhibition than planktonic cells. The minimum bactericidal concentrations (MBCs) of nanosilver (size from 15 to 21 nm), defined as the lowest concentration that kills at least 99.9% of a planktonic or biofilm bacterial population, were 38 and 10 mg/L Ag, respectively. For comparison, silver ions were more toxic to E. coli than nanosilver with MBCs of 2.4 and 1.2 mg/L Ag for planktonic and biofilm cultures, respectively. Nanosilver was aggregated in the presence of planktonic or biofilm-forming cells resulting in an increase of average particle size by a factor of 15 and 40, respectively. The nanosilver particles were able to penetrate to approximately 40 μm in a thick biofilm after 1-h exposure. These findings suggested that biofilm resistance to nanosilver could be at least partially due to nanoparticle aggregation and retarded silver ion/particle diffusion.     

Drinking water and biofilm disinfection by Fenton-like reaction

A Fenton-like disinfection process was conducted with Fenton's reagent (H₂O₂) at pH 3 or 5 on autochthonous drinking water biofilms grown on corroded or non-corroded pipe material. The biofilm disinfection by Fenton-like oxidation was limited by the low content of iron and copper in the biomass grown on non-corroded plumbing. It was slightly improved by spiking the distribution system with some additional iron source (soluble iron II or ferrihydrite particles appeared as interesting candidates). However successful in situ disinfection of biofilms was only achieved in fully corroded cast iron pipes using H₂O₂ and adjusting the pH to 5. These new results provide additional support for the use of Fenton's processes for cleaning drinking water distribution systems contaminated with biological agents or organics.     

Reversible shift in the α-, β- and γ-proteobacteria populations of drinking water biofilms during discontinuous chlorination

As disinfection strategies could support a shift of some bacterial populations, the biodiversity of drinking water biofilms depending on the disinfectant concentrations was explored. The effect of different chlorine sequences applied for several weeks (0.1-0.4-0.1 mg Cl₂ L⁻¹ or vice versa) was tested on the abundance of the α-, β- and γ-proteobacteria populations, used as indicators of changes in bacterial populations within drinking water biofilms. Using dynamic (industrial pilot) and batch (bench scale) conditions, our work demonstrated the ability of the 3 proteobacteria subclasses to re-organize following discontinuous chlorinations. The β- and γ-proteobacteria subclasses were favoured by high free residual chlorine concentrations (0.4 mg Cl₂ L⁻¹) while α-proteobacteria population was sensitive to this oxidant level. The proteobacteria population shifts within the biofilm exposed to discontinuous chlorination were reversible. The resilience of the biofilm proteobacteria populations exposed to oxidant stress questioned the emergence of bacterial population less sensitive to chlorine.     

Mechanisms of Escherichia coli inactivation by several disinfectants

The objective of this study was to elucidate dominant mechanisms of inactivation, i.e. surface attack versus intracellular attack, during application of common water disinfectants such as ozone, chlorine dioxide, free chlorine and UV irradiation. Escherichia coli was used as a representative microorganism. During cell inactivation, protein release, lipid peroxidation, cell permeability change, damage in intracellular enzyme and morphological change were comparatively examined. For the same level of cell inactivation by chemical disinfectants, cell surface damage was more pronounced with strong oxidant such as ozone while damage in inner cell components was more apparent with weaker oxidant such as free chlorine. Chlorine dioxide showed the inactivation mechanism between these two disinfectants. The results suggest that the mechanism of cell inactivation is primarily related to the reactivity of chemical disinfectant. In contrast to chemical disinfectants, cell inactivation by UV occurred without any changes measurable with the methods employed. Understanding the differences in inactivation mechanisms presented herein is critical to identify rate-limiting steps involved in the inactivation process as well as to develop more effective disinfection strategies.     

Sequential UV- and chlorine-based disinfection to mitigate Escherichia coli in drinking water biofilms

This study was designed to examine the potential downstream benefits of sequential disinfection to control the persistence of Escherichia coli under conditions relevant to drinking water distribution systems. Eight annular reactors (four polycarbonate and four cast iron) were setup in parallel to address various factors that could influence biofilm growth in distribution systems. Eight reactors were treated with chlorine, chlorine dioxide and monochloramine alone or in combination with UV to examine the effects on Escherichia coli growth and persistence in both the effluent and biofilm. In general, UV-treated systems in combination with chlorine or chlorine dioxide and monochloramine achieved greater log reductions in both effluent and biofilm than systems treated with chlorine-based disinfectants alone. However, during UV-low chlorine disinfection, E. coli was found to persist at low levels, suggesting that the UV treatment had instigated an adaptive mutation. During UV-chlorine-dioxide treatment, the E. coli that was initially below the detection limit reappeared during a low level of disinfection (0.2 mg/L) in the cast iron systems. Chloramine was shown to be effective in disinfecting suspended E. coli in the effluent but was unable to reduce biofilm counts to below the detection limit. Issues such as repair mechanism of E. coli and nitrification could help explain some of these aberrations. Improved understanding of the ability of chlorine-based disinfectant in combination with UV to provide sufficient disinfection will ultimately effect in improved management and safety of drinking water.     

Probing young drinking water biofilms with hard and soft particles

The aim of our study was to investigate, through the use of soft (Escherichia coli) and hard (polystyrene microspheres) particles, the distribution and persistence of allochthonous particles inoculated in drinking water flow chambers. Biofilms were allowed to grow for 7-10 months in tap water from Nancy's drinking water network and were composed of bacterial aggregates and filamentous fungi. Both model particles adhered almost exclusively on the biofilms (i.e. on the bacterial aggregates and on the filamentous structures) and not directly on the uncolonized walls (glass or Plexiglas). Biofilm age (i.e. bacterial density and biofilm properties) and convective-diffusion were found to govern particle accumulation: older biofilms and higher wall shear rates both increased the velocity and the amount of particle deposition on the biofilm. Persistence of the polystyrene particles was measured over a two-month period after inoculation. Accumulation amounts were found to be very different between hard and soft particles as only 0.03‰ of the soft particles inoculated accumulated in the biofilm against 0.3-0.8% for hard particles.     

Evaluation of some halogen biocides using a microbial biofilm system

A simple method for the formation of microbial biofilms of three species, Pseudomonas fluorescens, Pseudomonas aeruginosa, and Klebsiella pneumoniae, on a small glass slide was established, and its suitability for evaluation of disinfectant efficacy was examined. The biofilms formed were observed in situ by confocal laser scanning microscopy (CLSM). Using the biofilms established, biocidal efficacy of several halogen biocides, such as hypochlorite (HOCl), bromochlorodimethylhydantoin (Br, Cl-DMH), ammonia monochloramine (NH2Cl), a stabilized hypobromite biocide named STABREX, and a mixed solution of NH4Br and HOCl, was evaluated. The formation of NHBrCl in the mixed solution was indicated by UV spectra analysis. Biofilm cells were more resistant to these biocides than planktonic cells and the extent of resistance varied with the biocide tested. Among the biocides tested, the biocidal potency of HOCl was the most susceptible to the change brought about by biofilm formation. By CLSM observation, differences in biofilm conformation were revealed between the microbial species. The efficacy of the biocide tested varied with the structure of biofilms formed. The assay method developed in the present study would be useful for further investigation on biofilm disinfection.     

Synergistic effect of the sequential use of UV irradiation and chlorine to disinfect reclaimed water

The effectiveness of UV and chlorination, used individually and sequentially, was investigated in killing pathogenic microorganisms and inhibiting the formation of disinfection by-products in two different municipal wastewaters for the source water of reclaimed water, which were from a microfilter (W1) and membrane bioreactor (W2) respectively. Heterotrophic plate count (HPC), total bacteria count (TBC), and total coliform (TC) were selected to evaluate the efficiency of different disinfection processes. UV inactivation of the three bacteria followed first-order kinetics in W1 wastewater, but in W2 wastewater, the UV dose-response curve trailed beyond approximately 10 mJ/cm² UV. The higher number of particles in the W2 might have protected the bacteria against UV damage, as UV light alone was not effective in killing HPC in W2 wastewater with higher turbidity. However, chlorine was more effective in W2 than in W1 for the three bacteria inactivation owing to the greater formation of inorganic and organic chloramines in W1 wastewater. Complete inactivation of HPC in W1 wastewater required a chlorine dose higher than 5.5 mg/L, whereas 4.5 mg/L chlorine gave the equivalent result in W2 wastewater. In contrast, sequential UV and chlorine treatment produced a synergistic effect in both wastewater systems and was the most effective option for complete removal of all three bacteria. UV disinfection lowered the required chlorine dose in W1, but not in W2, because of the higher chlorine consumption in W2 wastewater. However, UV irradiation decreased total trihalomethane formation during chlorination in both wastewaters.     

Survival of Mycobacterium avium in a model distribution system

A pilot study was designed to examine the impact of nutrient levels, pipe materials, and disinfection on the survival of M. avium in model drinking water distribution system biofilms. Studies showed that the survival of the organism was dependant upon a complex interaction between pipe surface, nutrient levels, and disinfectants. The findings showed that when no disinfection was applied, M. avium could be recovered from biofilms at nutrient levels of 50microg/L assimilable organic carbon. M. avium concentrations were lower on copper pipe surfaces following disinfection with free chlorine as compared to monochloramine. However, due to the interference of corrosion products, chloramination of iron pipe surfaces controlled M. avium levels better than free chlorine. These data demonstrate the significance of pipe materials on the survival of M. avium complex in biofilms. Elimination of competitive heterotrophic bacteria on copper pipe surfaces by the application of disinfection resulted in a population of nearly 100% M. avium. Heat treatment of M. avium biofilms was affected by the pipe composition and organic content of the water. Effluent temperatures >53 degrees C were required to control the occurrence of M. avium in the pipeline system. Although additional studies are required using improved detection methods, the results of this investigation suggest that reducing the biodegradable organic material in drinking water, control of corrosion, maintenance of an effective disinfectant residual, and management of hot water temperatures can help limit the occurrence of M. avium complex in drinking water biofilms.     

Investigating synergism during sequential inactivation of MS-2 phage and Bacillus subtilis spores with UV/H2O2 followed by free chlorine

A sequential application of UV as a primary disinfectant with and without H₂O₂ addition followed by free chlorine as secondary, residual disinfectant was performed to evaluate the synergistic inactivation of selected indicator microorganisms, MS-2 bacteriophage and Bacillus subtilis spores. No synergism was observed when the UV irradiation treatment was followed by free chlorine, i.e., the overall level of inactivation was the same as the sum of the inactivation levels achieved by each disinfection step. With the addition of H₂O₂ in the primary UV disinfection step, however, enhanced microbial inactivation was observed. The synergism was observed in two folds manners: (1) additional inactivation achieved by hydroxyl radicals generated from the photolysis of H₂O₂ in the primary UV disinfection step, and (2) damage to microorganisms in the primary step which facilitated the subsequent chlorine inactivation. Addition of H₂O₂ in the primary disinfection step was also found to be beneficial for the degradation of selected model organic pollutants including bisphenol-A (endocrine disruptor), geosmin (taste and odor causing compound) and 2,4-D (herbicide). The results suggest that the efficiency of UV/free chlorine sequential disinfection processes, which are widely employed in drinking water treatment, could be significantly enhanced by adding H₂O₂ in the primary step and hence converting the UV process to an advanced oxidation process.     

Norovirus and MS2 inactivation kinetics of UV-A and UV-B with and without TiO2

Germicidal ultraviolet, such as 254-nm UV-C, is a common method of disinfection of pathogenic enteric viruses. However, the disinfection efficacies of UV-A or -B in terms of inactivating waterborne viruses such as norovirus have not been characterized. We evaluated the inactivation kinetics of MS2 bacteriophage and murine norovirus (MNV), a surrogate of human norovirus (NoV), by UV-A and -B. In addition to UV disinfection, we further investigated whether the presence of TiO₂ could enhance the virus inactivation kinetics of UV-A and -B. Both MS2 and MNV were highly resistant to UV-A. However, the addition of TiO₂ enhanced the efficacy of UV-A for inactivating these viruses. UV-A dose of 1379 mJ/cm² resulted in a 4 log₁₀ reduction. In comparison, UV-B alone effectively inactivated both MS2 and MNV, as evidenced by the 4 log₁₀ reduction by 367 mJ/cm² of UV-B. The addition of TiO₂ increased the inactivation of MS2; however, it did not significantly increase the efficacy of UV-B disinfection for inactivating MNV. When these treatments were applied to field water such as groundwater, the results were generally consistent with the laboratory findings. Our results clearly indicated that UV-B is useful for the disinfection of waterborne norovirus. However, MNV was quite resistant to UV-A, and UV-A effectively inactivated the tested viruses only when used in combination with TiO₂.     

Effects of silver nanoparticles on wastewater biofilms

The goal of this research is to understand the potential antibacterial effect of silver nanoparticles (Ag-NPs) on biological wastewater treatment processes. It was found that original wastewater biofilms are highly tolerant to the Ag-NP treatment. With an application of 200 mg Ag/L Ag-NPs, the reduction of biofilm bacteria measured by heterotrophic plate counts was insignificant after 24 h. After the removal of loosely bound extracellular polymeric substances (EPS), the viability of wastewater biofilms was reduced when treated under the same conditions. By contrast, when treated as planktonic pure culture, bacteria isolated from the wastewater biofilms were highly vulnerable to Ag-NPs. With a similar initial cell density, most bacteria died within 1 h with the application of 1 mg Ag/L Ag-NPs. The results obtained here indicate that EPS and microbial community interactions in the biofilms play important roles in controlling the antimicrobial effects of Ag-NPs. In addition, slow growth rates may enhance the tolerance of certain bacteria to Ag-NPs. The effects of Ag-NPs on the entire microbial community in wastewater biofilms were analyzed using polymerase chain reaction-denaturing gradient gel electrophoresis, PCR-DGGE. The studies showed that the microbial susceptibility to Ag-NPs is different for each microorganism. For instance, Thiotrichales is more sensitive to Ag-NPs than other biofilm bacteria.     

The assessment of particle association and UV disinfection of wastewater using indigenous spore-forming bacteria

Studies have shown that association between particles and coliform bacteria in wastewater influence the inactivation of these microorganisms by ultraviolet (UV) irradiation. This research investigated the potential use of indigenous aerobic spore-forming (ASF) bacteria for studying the particle - microorganism interaction and its effect on UV disinfection of protozoan pathogens, such as Giardia spp. and Cryptosporidium spp., present in effluents from full-scale municipal wastewater treatment plants. The effect of particle - ASF association was determined by homogenizing wastewater effluent samples before and after exposure to controlled UV doses delivered by a bench-scale collimated beam apparatus. Particle association between Bacillus subtilis spores added to wastewater and wastewater particles was also assessed. The results indicate that spores are not significantly associated with wastewater particulate matter and particle association does not significantly affect the inactivation of indigenous spores present in wastewater by UV radiation in this study.     

Species association increases biofilm resistance to chemical and mechanical treatments

The study of biofilm ecology and interactions might help to improve our understanding of their resistance mechanisms to control strategies. Concerns that the diversity of the biofilm communities can affect disinfection efficacy have led us to examine the effect of two antimicrobial agents on two important spoilage bacteria. Studies were conducted on single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens. Biofilms were formed on a stainless steel rotating device, in a bioreactor, at a constant Reynolds number of agitation (Re_A). Biofilm phenotypic characterization showed significant differences, mainly in the metabolic activity and both extracellular proteins and polysaccharides content. Cetyl trimethyl ammonium bromide (CTAB) and glutaraldehyde (GLUT) solutions in conjunction with increasing Re_A were used to treat biofilms in order to assess their ability to kill and remove biofilms. B. cereus and P. fluorescens biofilms were stratified in a layered structure with each layer having differential tolerance to chemical and mechanical stresses. Dual species biofilms and P. fluorescens single biofilms had both the highest resistance to removal when pre-treated with CTAB and GLUT, respectively. B. cereus biofilms were the most affected by hydrodynamic disturbance and the most susceptible to antimicrobials. Dual biofilms were more resistant to antimicrobials than each single species biofilm, with a significant proportion of the population remaining in a viable state after exposure to CTAB or GLUT. Moreover, the species association increased the proportion of viable cells of both bacteria, comparatively to the single species scenarios, enhancing each other's survival to antimicrobials and the biofilm shear stress stability.     

Determination of nutrients limiting biofilm formation and the subsequent impact on disinfectant decay.

Understanding the contribution of both organic and inorganic nutrients to biofilm development and the subsequent impact of developed biofilms on disinfectant decay are important requirements for distribution system management strategies. Nutrient limitation may be one way to control biofilm development without increasing disinfectant dosing. Little is known, however, of the nutrient requirements of biofilms in distribution systems. Indeed, the effects on biofilm development due to the addition of nutrients to distribution systems and what impact biofilm development may have on disinfectant decay is still poorly understood. This study used annular reactors to determine the nutrients limiting for biofilm development in drinking water from two different Sydney sources and the subsequent effects of biofilm development on disinfectant decay. It was found that biofilm development in Sydney water was limited by organic carbon and that biofilm development promoted chloramine decay. Moreover, biofilm development occurred in the presence of chloramine. The ability of biofilms to respond to increases in disinfectant concentrations was dependent on the biomass of the biofilms. In a comparative study using chlorinated drinking water containing very low levels of organic carbon, biofilm development was not detected. Removal of organic carbon resulted in greater persistence of chlorine, which led to greater biofilm control. It was also shown that biofilms could contribute cells to the aqueous phase. The results of the study indicate that treatment and system management strategies should incorporate organic carbon removal to limit biofilm development through a combination of retarding bacterial growth and enhancing disinfectant persistence.     

Protection against UV disinfection of E. coli bacteria and B. subtilis spores ingested by C. elegans nematodes

Nematodes, which occur abundantly in granular media filters of drinking water treatment plants and in distribution systems, can ingest and transport pathogenic bacteria and provide them protection against chemical disinfectants. However, protection against UV disinfection had not been investigated to date.In this study, Caenorhabditis elegans nematodes (wild-type strain N2) were allowed to feed on Escherichia coli OP50 and Bacillus subtilis spores before being exposed to 5 and 40 mJ/cm² UV fluences, using a collimated beam apparatus (LP, 254 nm). Sonication (15 W, 60 s) was used to extract bacteria from nematode guts following UV exposure in order to assess the amount of ingested bacteria that resisted the UV treatment using a standard culture method. Bacteria located inside the gut of C. elegans were shown to benefit from a significant protection against UV. Approximately 15% of the applied UV fluence of 40 mJ/cm² (as typically used in WTP) was found to reach the bacteria located inside nematode guts based on the inactivation of recovered bacteria (2.7 log reduction of E. coli bacteria and 0.7 log reduction of B. subtilis spores at 40 mJ/cm²). To our knowledge, this study is the first demonstration of the protection effect of bacterial internalization by higher organisms against UV treatment, using the specific case of E. coli and B. subtilis spores ingested by C. elegans.