Biofilm Sloughing Research Articles

A poroelastic [formula omitted]-SPH model for modeling biofilm deformation and sloughing in microfluidic channels

We present a poroelastic fluid–structure interaction δ-SPH model for simulating biofilm deformation and sloughing in microfluidic channels. Based on the mixture theory, this model accurately handles large deformations of solid structures and simultaneously simulates seepage flow within the porous biofilm and external flow. A key advantage of the model is its reliance on the solid phase material properties rather than the averaged properties of the biofilm, enabling the modeling of changes in porosity and biofilm material properties induced by loads. Model verification is achieved through two benchmark problems, followed by application to biofilm deformation and sloughing studies. The Young’s modulus of biofilms is a key parameter of interest in many studies. Calibration of the biofilm’s solid material elasticity modulus by using the presented model yields a value of 200 Pa, aligning with previous reports in literature. Additionally, the model is capable of simulating the sloughing process by evaluating local von Mises equivalent stress, reproducing different detachment patterns corresponding to various material strengths. This δ-SPH model offers an efficient numerical tool for biofilm analysis and is extendable to simulate other fluid–structure interaction problems involving porous media with large deformations, such as soil, debris flow, and biological tissues.

Micrococcal nuclease regulates biofilm formation and dispersal in methicillin-resistant Staphylococcus aureus USA300.

Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA that hold the biofilm together. MRSA cells secrete micrococcal nuclease (Nuc1), which degrades double-stranded DNA. In this study, we used standard methodologies to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. We quantified biofilm formation and extracellular DNA (eDNA) levels in broth and agar cultures. In some experiments, cultures were supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods and enumerating detached colony-forming units (CFUs), and biofilm sloughing was investigated by perfusing biofilms cultured in glass tubes with fresh broth and measuring the sizes of the detached cell aggregates. We found that an MRSA nuc1- mutant strain produced significantly more biofilm and more eDNA than a wild-type strain, both in the absence and presence of sub-MIC amoxicillin. nuc1- mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or complementing the nuc1- mutant. In the sloughing assay, nuc1- mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.IMPORTANCEInfections caused by antibiotic-resistant bacteria known as methicillin-resistant Staphylococcus aureus (MRSA) are a significant problem in hospitals. MRSA forms adherent biofilms on implanted medical devices such as catheters and breathing tubes. Bacteria can detach from biofilms on these devices and spread to other parts of the body such as the blood or lungs, where they can cause life-threatening infections. In this article, researchers show that MRSA secretes an enzyme known as thermonuclease that causes bacteria to detach from the biofilm. This is important because understanding the mechanism by which MRSA detaches from biofilms could lead to the development of procedures to mitigate the problem.

Evaluating scaling of capillary photo-biofilm reactors for high cell density cultivation of mixed trophies artificial microbial consortia.

Capillary biofilm reactors (CBRs) are attractive for growing photoautotrophic bacteria as they allow high cell-density cultivation. Here, we evaluated the CBR system's suitability to grow an artificial consortium composed of Synechocystis sp. PCC 6803 and Pseudomonas sp. VBL120. The impact of reactor material, flow rate, pH, O2, and medium composition on biomass development and long-term biofilm stability at different reactor scales was studied. Silicone was superior over other materials like glass or PVC due to its excellent O2 permeability. High flow rates of 520μLmin-1 prevented biofilm sloughing in 1m capillary reactors, leading to a 54% higher biomass dry weight combined with the lowest O2 concentration inside the reactor compared to standard operating conditions. Further increase in reactor length to 5m revealed a limitation in trace elements. Increasing trace elements by a factor of five allowed for complete surface coverage with a biomass dry weight of 36.8gm-2 and, thus, a successful CBR scale-up by a factor of 25. Practical application: Cyanobacteria use light energy to upgrade CO2, thereby holding the potential for carbon-neutral production processes. One of the persisting challenges is low cell density due to light limitations and O2 accumulation often occurring in established flat panel or tubular photobioreactors. Compared to planktonic cultures, much higher cell densities (factor 10 to 100) can be obtained in cyanobacterial biofilms. The capillary biofilm reactor (CBR) offers good growth conditions for cyanobacterial biofilms, but its applicability has been shown only on the laboratory scale. Here, a first scale-up study based on sizing up was performed, testing the feasibility of this system for large-scale applications. We demonstrate that by optimizing nutrient supply and flow conditions, the system could be enlarged by factor 25 by enhancing the length of the reactor. This reactor concept, combined with cyanobacterial biofilms and numbering up, holds the potential to be applied as a flexible, carbon-neutral production platform for value-added compounds.

Pollutant removal performance and microbial responses of pure moving bed biofilm reactor to the successional sulfadiazine exposure

Pure Moving bed biofilm reactor (pure MBBR) with biofilm as biomass core is a viable process for treating antibiotics. The influences of sulfadiazine (SDZ) on pure MBBR have yet to be investigated in depth. This study examined the responses of biofilm exposure to different doses of SDZ (0, 1, 3, 5, 7 and 10 mg/L) in the pure MBBR. Under continuous-flow conditions, pure MBBR held a high SDZ removal rate, but SDZ significantly inhibited COD and TN removal. However, 1–3 mg/L SDZ could enhance NH4+-N and TP removal. And these observations were confirmed by biomass analysis and multi-omics sequencing data. SDZ accelerated the sloughing and renewal of aerobic biofilm, thus shortening the solids residence time. The biofilm communities were strongly shifted by SDZ and the activity of autotrophs was less inhibited by SDZ than that of heterotrophs. Meanwhile, SDZ did not alter the biofilm's primary nitrogen metabolism mechanism, while disrupting the ratio of denitrification genes to nitrification genes. SDZ effectively promoted the gene expression of amoCAB and hao, but restrained norBC. The increased abundance of resistance genes Sul1–3 was a major reason for the biofilm could adapt to the high concentration of SDZ. Overall, the pure MBBR could operate well when SDZ was below 3 mg/L. This study provided new insight into antibiotic wastewater treatment through pure MBBR.

Micropollutant transformation and toxicity: Electrochemical ozonation versus biological metabolism

Environmental water sources are constantly polluted by anthropogenic compounds, not always minimized by conventional water treatment methods to remove these compounds at the micro- and nano-range. The absolute concentrations of a suite of seven representative environmental micropollutants were compared pre- and post-treatment with both ozone and microbial biofilms, in terms of removal efficiencies and toxicity assays. Both synthetic micropollutant mixes and environmental water samples were evaluated. The study started with two representative micropollutants (carbamazepine, CBZ, and sulfamethoxazole, SMX), and broadened into a suite of pollutants, evaluating whole-sample eco-toxicological footprints. An ozone concentration of 4.24 ± 0.27 mg/L in tap water, resulted in an 87.9% and 96.5% removal of CBZ and SMX, respectively, within 1 min. Despite almost immediate removal of parent micropollutants by oxidation, endocrine disruption potential (anti-estrogenicity) of CBZ and SMX required up to 240 min of ozone treatment to show no assay effect. A broader suite of micropollutants in more complex environmental matrices showed scavenging of ozone (2.95 ± 0.17–0.25 ± 0.03 mg/L) and varying micropollutant recalcitrance to oxidation. Lower matrix pollution led to lower reduction in eco-toxicity. Microbial degradation of CBZ and SMX (56% and 70% versus 19% and 79%, respectively, in duplicate biofilms) by nutrient-limited biofilms showed less removal than ozonation, with marked variation due to the stochastic nature of biofilm sloughing. Microbial degradation of CBZ and SMX resulted in an increase of >90% in both estrogenicity and Aliivibrio inhibition. The results obtained from this study address a gap in understanding the removal efficiency of micropollutants, where the removal process often receives more attention than the comparative reduction of toxicological effects. This shift from a controlled laboratory environment to real-world scenarios also provided comparative insights into the removal of micropollutants and the eco-toxicity of the transformation by-products of each process.

Critical shear stresses of Pseudomonas aeruginosa biofilms from dental unit waterlines studied using microfluidics and additional magnesium ions

A microfluidic approach was used to study the effect of shear stress on biofilms from a dental unit waterline (DUWL)-isolated P. aeruginosa strain, PPF-1. During the application of relevant shear stress levels to DUWLs, the response of the PPF-1 biofilm was observed and compared to that of a well-studied clinical P. aeruginosa strain, PAO1. The response measurements were repeated for biofilms exposed to additional Mg2+ ions. Optical density maps were transformed into pseudo three-dimensional representations of the complex biofilm structures, and computational fluid dynamic simulations were used to determine the critical shear stresses for biofilm sloughing. In the absence of Mg2+, PPF-1 biofilms showed weaker attachment than PAO1 biofilms and highly intertwined slough/regrowth cycles occurring within the shear stress range of 1.42 ± 0.32 and 0.95 ± 0.27 Pa. This suggests that in a low ionic environment, the PPF-1 strain produces ejected biofilm material nearly continuously, which can result in increased downstream colonization of engineered flow systems. Introducing Mg2+ into the PPF-1 biofilm culture increased mechanical stability, which resulted in elevated tolerances to shear stresses up to a critical value of 5.43 ± 1.52 Pa, which was similar to the critical shear stress value of 4.23 ± 1.22 Pa for the PAO1 strain. Moreover, the enhanced Mg2+ concentrations seemed to place the PPF-1 biofilm into a viscoplastic mechanical state, which resulted in signature responses to critical shear stresses, such as catastrophic sloughing involving abrupt tearing that produced clean edges at the fracture boundary, indicating that the biofilm had become brittle.

Fate of sloughed biomass in integrated fixed-film systems.

Fate of biofilm sloughing was assessed in a laboratory-scale (LS) integrated fixed-film sequencing batch reactor (IF-SBR) treating synthetic wastewater and in a full-scale (FS) integrated fixed-film activated sludge (IFAS) system treating municipal wastewater. It was observed that the properties of biofilms and flocs, including sludge volume index (SVI), mixed liquor suspended solids (MLSS), effluent suspended solids (ESS), relative hydrophobicity, and composition of extracellular polymeric substance (EPS) were associated with biofilm sloughing and formation of large granular flocs in the LS IF–SBR. In the FS IFAS system, the changes were studied at the molecular level. For example, the extracted EPS content results (the protein to polysaccharide ratio decreased in the flocs and increased in the biofilms, with biofilm sloughing) were complemented with the confocal laser scanning microscopy (CLSM) coupled with molecular specific staining. CLSM analyses revealed that micro-colonies rich in polysaccharides readily sloughed from the carriers. Live-dead staining revealed areas of the biofilm where the viability of biomass was a contributing factor associated with areas of the biofilm susceptible to sloughing. 16S rRNA gene sequencing (Illumina) of FS IFAS samples revealed greater diversity (α-diversity) in biofilms compared to flocs. Biofilm sloughing resulted in a decrease in diversity in biofilms and a corresponding increase in the flocs during sloughing. Microbial population dynamics revealed that bacteria known for denitrification (for example, Comamonadaceae) detached from the biofilms during sloughing, readily associated with the suspended biomass, and were retained in the bioreactors.

Effect of disinfectant exposure and starvation treatment on the detachment of simulated drinking water biofilms

Biofilms were one of the main habitats of microbes in the drinking water distribution system. The variation of environmental conditions can lead to the detachment of biofilms and the deterioration of water quality. In this study, the effects of disinfectant exposure and starvation treatment on the detachment of biofilms were investigated. The results showed that detaching rate increased with the concentration of chloramine in the inlet water and 1.0 mg/L of chloramine led to the largest detached biomass. The starvation treatment resulted in less biofilm biomass but the detaching rates of treated biofilms were higher than those without starvation. The 16S rRNA sequencing results showed that detached and stubborn biofilms had a significant difference in microbial diversity and richness. The microbial community composition of the two types of biofilm showed the difference in the abundance of Nitrospira, Bryobacter, Hyphomicrobium, and Pedomicrobium. Chloramine exposure did not have a significant impact on the microbial community while the starvation treatment led to a higher abundance of chemolithotrophs bacteria. Metagenomic results indicated that detached biofilms had higher abundances of ARGs and starvation treatment could enrich the ARGs. The results of this research could provide the knowledge of biofilm sloughing and help understand the health risk of antibiotic resistance in drinking water.

Effects of cations on biofilm formation and characteristics in integrated fixed film activated sludge process at different carbon and nitrogen loadings

Both divalent cations including calcium and magnesium play important roles for microbial aggregates in binding to negatively charged functional groups on bacterial surfaces, in extracellular polymeric substances (EPS), and on inorganic materials in flocs and biofilms. Monovalent cations such as sodium and potassium deteriorate the floc structure and physical properties. The Integrated Fixed Activated Sludge (IFAS) process employs fixed film media in the aerobic zone; therefore, both monovalent and divalent cations are involved in the process performances. In this study, the effects of cations indicated as the monovalent to divalent cations (M/D) ratio on the biofilm formation and characteristics, and on the IFAS performances for carbon and ammonium removals were evaluated. The experiments were conducted in three IFAS systems feeding with the same wastewater but different M/D ratios and two carbon and nitrogen loadings. The findings revealed that high monovalent with low divalent cations at the M/D ratios higher than 2.0 produced excessive polysaccharides in EPS resulting in high viscosity of activated sludge flocs causing viscous bulking with high SVI values, decreasing the biofilm formation, and increasing the biofilm sloughing. Increasing of both monovalent and carbon loading increased more polysaccharides in the EPS leading to the failures of IFAS system. Nitrification failed at higher M/D ratios because of less nitrifiers in flocs and biofilm. The M/D ratio less than 2.0 is suggested to minimize the excessive EPS production in the IFAS system, especially at high organic loading.

Treatment of low strength wastewater using compact submerged aerobic fixed film (SAFF) reactor filled with high specific surface area synthetic media.

Studies on laboratory-scale submerged aerobic fixed film reactor (SAFF) packed with synthetic media having specific surface area of 165 m2/m3 with a void volume of 89% were carried out to assess its performance under various organic loading rates (OLR) and hydraulic retention times (HRT). Synthetic wastewater having chemical oxygen demand (COD) and biochemical oxygen demand (BOD) of 400 ± 10% and 210 ± 10% mg/L respectively was fed and the reactor was subjected to OLRs ranging from 0.37 to 1.26 kg COD/m3.d. It was observed that steady sloughing of biofilm occurs within the SAFF reactor all the times and average concentration of sloughed biomass in the effluent was 26 mg/L. The COD and BOD removal efficiencies varied between 85 and 89% and 86 to 94%, respectively. The kinetic studies demonstrated that SAFF reactor followed Stover-Kincannon and Grau models, with high correlation coefficients (R2) of 0.9977 and 0.9916, respectively. Thus, the values of kinetic coefficients such as maximum substrate utilization rate, Umax = 64.1 g/(L.d); saturation value constant, KB = 72.31 g/(L.d) and Grau second-order substrate removal rate constant, Ks = 2.44 day-1 can be useful to develop and design large scale SAFF reactors. Finally, the study reveals that the optimum range for OLR can vary within 0.68-0.94 kg COD/m3.d.

Predation creates unique void layer in membrane-aerated biofilms

The membrane-aerated biofilm reactor (MABR) is a novel wastewater treatment technology based on oxygen-supplying membranes. The counter diffusion of oxygen and electron donors in MABRs leads to unique behavior, and we hypothesized it also could impact predation. We used optical coherence tomography (OCT), microsensor analyses, and mathematical modeling to investigate predation in membrane-aerated biofilms (MABs). When protozoa were excluded from the inoculum, the MAB's OCT-observable void fraction was around 5%. When protozoa were included, the void fraction grew to nearly 50%, with large, continuous voids at the base of the biofilm. Real-time OCT imaging showed highly motile protozoa in the voids. MABs with protozoa and a high bulk COD (270 mg/L) only had 4% void fraction. DNA sequencing revealed a high relative abundance of amoeba in both high and low-COD MABs. Flagellates were only abundant in the low-COD MAB. Modeling also suggested a relationship between substrate concentrations, diffusion mode (co- or counter-diffusion), and biofilm void fraction. Results suggest that amoeba proliferate in the biofilm interior, especially in the aerobic zones. Voids form once COD limitation at the base of MABs allows predation rates to exceed microbial growth rates. Once formed, the voids provide a niche for motile protozoa, which expand the voids into a large, continuous gap. This increases the potential for biofilm sloughing, and may have detrimental effects on slow-growing, aerobic microorganisms such as nitrifying bacteria.

Pilot scale study of sequencing batch reactor (SBR) retrofit with integrated fixed film activated sludge (IFAS): nitrogen removal and design consideration

IFAS process was coupled with SBR operation in a pilot-scale reactor to verify the feasibility and to evaluate the performance of IFAS-SBR. Significant nitrification improvement in the IFAS-SBR system was observed, which is attributed to both the introduction of attached-growth biomass on media carriers and the “seeding effect” by biofilm sloughing.

Decentralized wastewater treatment using ‘Pumped Flow Biofilm Reactor’ (PFBR) technology

Clean water resources are imperative for sustainable development. Thus, protection and management of waters receiving wastewater discharges have received significant attention from policy and regulatory bodies. The quality of wastewater effluent must meet regional (e.g. Water Framework Directive), national and local discharge standards. In addition, there is now significant pressure on engineers and operators to reduce energy consumption, sludge production and operation/maintenance issues, particularly at small-scale and decentralized wastewater facilities. Therefore, significant interest has risen in new technologies and operational insights which can (i) minimize operating costs; (ii) simplify and reduce the use of mechanical equipment; (iii) result in low sludge production; and (iv) ease operation/maintenance. This study investigated the performance of a small-scale municipal wastewater facility over 5 months from commissioning. The facility uses a new biofilm-based technology – the pumped flow biofilm reactor. Two experimental periods Phase 1 (28 to 36 days) and Phase 2 (Days 100 to 146) were examined. During Phase 2, removal rates averaged 98% for 5-day biochemical oxygen demand (BOD5), 93% for total suspended solids, and 94% ammoniacal-nitrogen (NH4-N). Energy requirements averaged 0.22 kWh.m treated−3 and 1.74 kWh.kg-BOD5 removed−1. Extensive, camera-based studies revealed minimal excess sludge in the reactor tanks and sludge removal was not required during the study period. The use of vertically stacked plastic media to support the biofilm may have limited biofilm sloughing. Sludge yield during steady state operation was estimated at around 0.03 g-SS.g-COD removed−1. The study indicates that given careful design and operation, small-scale wastewater treatment systems can be as efficient as much larger, fully manned plants.

Effect of increasing organic loading rates on the performance of moving-bed biofilm reactors filled with different support media: Assessing the activity of suspended and attached biomass fractions

In this study, two moving-bed biofilm reactors (MBBR1 and MBBR2) filled with different carrier media (Kaldnes K1 and Mutag Biochip, respectively) were subjected to increasing organic loading rates for 700 days. Regardless of the carrier used, both systems could withstand high organic loads up to 3.2kgCOD/(m3d), condition under which complete ammonium removal was achieved. However, the type of media influenced the quantity and distribution of attached biomass in the support, which in turn affected the activity of specific microbial functional groups in the biofilm. As the chemical oxygen demand (COD) input was gradually increased, the biofilm got thicker and the surface detachment rates were enhanced. Consequently, the amount of suspended solids has increased considerably to levels commonly found in hybrid bioreactors. Activity batch tests have shown that the contribution of the bulk phase biomass to the overall nitrification was very significant, being more relevant as the biofilm sloughing events became more intense. At constant organic loading rate, the hydraulic retention time (HRT) had a noticeable impact on the nitrification process, as it directly influenced the fraction of ammonium oxidized either by the attached or suspended biomass. Total nitrogen removal amounted up to 86 and 73% in MBBR1 and MBBR2, respectively.

Physicochemical characteristics and microbial community evolution of biofilms during the start-up period in a moving bed biofilm reactor

This study aimed to investigate biofilm properties evolution coupled with different ages during the start-up period in a moving bed biofilm reactor system. Physicochemical characteristics including adhesion force, extracellular polymeric substances (EPS), morphology as well as volatile solid and microbial community were studied. Results showed that the formation and development of biofilms exhibited four stages, including (I) initial attachment and young biofilm formation, (II) biofilms accumulation, (III) biofilm sloughing and updating, and (IV) biofilm maturation. During the whole start-up period, adhesion force was positively and significantly correlated with the contents of EPS, especially the content of polysaccharide. In addition, increased adhesion force and EPS were beneficial for biofilm retention. Gram-negative bacteria mainly including Sphaerotilus, Zoogloea and Haliscomenobacter were predominant in the initial stage. Actinobacteria was beneficial to resist sloughing. Furthermore, filamentous bacteria were dominant in maturation biofilm.

Microbial biogeography across a full-scale wastewater treatment plant transect: evidence for immigration between coupled processes

Wastewater treatment plants use a variety of bioreactor types and configurations to remove organic matter and nutrients. Little is known regarding the effects of different configurations and within-plant immigration on microbial community dynamics. Previously, we found that the structure of ammonia-oxidizing bacterial (AOB) communities in a full-scale dispersed growth activated sludge bioreactor correlated strongly with levels of NO2 (-) entering the reactor from an upstream trickling filter. Here, to further examine this puzzling association, we profile within-plant microbial biogeography (spatial variation) and test the hypothesis that substantial microbial immigration occurs along a transect (raw influent, trickling filter biofilm, trickling filter effluent, and activated sludge) at the same full-scale wastewater treatment plant. AOB amoA gene abundance increased >30-fold between influent and trickling filter effluent concomitant with NO2 (-) production, indicating unexpected growth and activity of AOB within the trickling filter. Nitrosomonas europaea was the dominant AOB phylotype in trickling filter biofilm and effluent, while a distinct "Nitrosomonas-like" lineage dominated in activated sludge. Prior time series indicated that this "Nitrosomonas-like" lineage was dominant when NO2 (-) levels in the trickling filter effluent (i.e., activated sludge influent) were low, while N. europaea became dominant in the activated sludge when NO2 (-) levels were high. This is consistent with the hypothesis that NO2 (-) production may cooccur with biofilm sloughing, releasing N. europaea from the trickling filter into the activated sludge bioreactor. Phylogenetic microarray (PhyloChip) analyses revealed significant spatial variation in taxonomic diversity, including a large excess of methanogens in the trickling filter relative to activated sludge and attenuation of Enterobacteriaceae across the transect, and demonstrated transport of a highly diverse microbial community via the trickling filter effluent to the activated sludge bioreactor. Our results provide compelling evidence that substantial immigration between coupled process units occurs and may exert significant influence over microbial community dynamics within staged bioreactors.

Organic removal activity in biofilm and suspended biomass fractions of MBBR systems

The moving bed biofilm reactor (MBBR) wastewater treatment process is usually designed based on the assumption that all activity in the process occurs in the biofilm on the MBBR carriers, although there is always some active biomass in the bulk liquid due to biofilm sloughing and, sometimes, free-growing bacteria. In this study the removal of organic matter is evaluated in laboratory-scale MBBR reactors under varying load, hydraulic retention time (HRT), oxygen concentration and volumetric filling degree of carriers in order to determine the heterotrophic activity in the different fractions of the MBBR biomass. The results showed that the heterotrophic conversions in an MBBR can show the same type of diffusion limited dependency on oxygen as nitrification, even for easily degradable substrates such as acetate. The contribution to the removal from the suspended biomass is shown to vary depending on HRT, as the amount of suspended solids changes. The developed method in this report is a useful tool for determining heterotrophic activity in the separate fractions of biomass in MBBRs.

The Impact of Biofilm Growth on Transport of Escherichia coli O157:H7 in Sand

Understanding the transport behavior, survival, and persistence of pathogens such as Escherichia coli O157:H7 in the subsurface is essential to protection of public health. In this study, the transport of E. coli O157:H7 in a two-dimensional bench-scale sand aquifer system, hereafter referred to as the sandbox, was investigated, with a focus on the impact of biofilm development on E. coli retention and survival. Biofilm growth was initiated through flushing with unsterilized groundwater and addition of glucose, nitrate, and phosphate. Retention of E. coli from an injection test in clean sand, prior to promotion of biofilm growth, was approximately 9%. Subsequent to biofilm growth, 47% of injected E. coli cells were retained under similar flow conditions. After 10 d of no flow, sterile water was flushed through the biofouled sandbox and substantial concentrations (up to 1.5 × 10(5) cells/mL) of E. coli were measured in the effluent indicating that E. coli had survived the starvation period. Confocal laser scanning microscopy revealed that E. coli were located not only on the surface but also within the biofilm. Imposition of starvation conditions resulted in biofilm sloughing and possible mobilization of biofilm-associated E. coli.

Biofilm sloughing and CBP formation in a 12-km transport system carrying chlorinated saline secondary effluent

A 12-km conveyance system has been placed in service for 10 years to transport treated municipal saline effluent which was not chlorinated. Now the effluent is being considered for chlorine disinfection to reduce Escherichia coli count to less than 1,000 cfu/100 mL. Since the system was not designed to carry the chlorinated flow, a simulator study has been conducted with a 68-m pipe loop (2.5“ ID) to assess the biofilm growth and sloughing when the effluent is subjected to chlorination. In addition, the potential formations of THM and HAA5 in the transport system are also assessed. Experimental results indicate that the level of biofilm growth in the pipe wall is mainly dictated by the organic strength of the effluent flow. For conveying the saline secondary effluent with a BOD of about 10 mg/L, the biofilm growth is approximately 160 μm. With chlorination of up to 5.0 mg/L, the biofilm growth and sloughing are not affected. As for CBP productions, 5.0 mg/L chlorination of the activated sludge effluent (w...

Evaluating aerobic endospores as indicators of intrusion in distribution systems

Aerobic endospores are naturally found in soil, easy to measure, and more resistant to chlorine than bacterial indicators are. The objective of this study was to assess whether aerobic endospores could be used as an indicator of intrusion in a full‐scale distribution system. The background aerobic endospore concentration in distributed water was low (average of 0.13 cfu/100 mL). Pipe deposits and biofilm sloughing were not found to be significant sources of endospores; concentrations in 8 of 10 spot‐flushing samples were not significantly different from those in distributed water. Only 5 of 71 water samples collected during flushing after main repairs showed endospore concentrations that were higher than they were during spotflushing. These observations suggest that aerobic endospores can be used as indicators of intrusion in distribution systems and could be useful to assess the adequacy of maintenance practices, identify areas of improvement, and manage incidents with risks of intrusion.