Effect of shear stress and growth conditions on detachment and physical properties of biofilms

Detachment is one of the major processes determining the physical structure and microbial functionalities of biofilms. To predict detachment, it is necessary to take the mechanical properties of the biofilm and the effect of both hydrodynamic and growth conditions into account. In this work, experiments were conducted with biofilms developed under various shear stresses and with various substrate natures. In addition, two cases were considered in order to differentiate between the effect of hydrodynamic factors and growth factors: the biofilms were directly grown under the targeted shear stress (τ) condition or they were precultivated under very low shear stress (0.01 Pa) and then exposed to high shear stress in the range of 0.1-13 Pa. An exponential and asymptotic decrease of the biofilm thickness and mass with increasing τ was observed in both cases. On contrary density, expressed as the biofilm dry mass on a known substratum divided by the average thickness increased with τ. Denitrifying biofilms always showed greater thickness and density than oxic biofilms. These results showed the presence of a compact basal layer that resisted shear stresses as high as 13 Pa whatever the culture conditions. Above this basal layer, the cohesion was lower and depended on the shear stress applied during biofilm development. The application of shear stress to the biofilms resulted in both detachment and compression, but detachment prevailed for the upper part of the biofilms and compression prevailed for the basal layers. A model of biofilm structure underlying the stratified character of this aggregate is given in terms of density and cohesion.     

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.     

Experimental toxicity and bioaccumulation of cadmium in freshwater periphytic diatoms in relation with biofilm maturity

A study was undertaken to examine cadmium accumulation in freshwater biofilm, its effects on biofilm development and on diatom community structure in laboratory experimental conditions. A suspension of a biofilm originated from the Riou-Mort River (South West France) was inoculated into three experimental units containing clean glass substrates under laboratory conditions. Settling and already developed biofilms were exposed to a Cd concentration of 100 µg L^− 1. Metal accumulation (total and intracellular metal content) in biofilms, dry weight and ash-free dry mass, diatom cell density and diatom community composition were analyzed. Both total and intracellular Cd accumulated by the biofilm throughout the experiment increased with duration of metal exposure. Biofilms in the course of maturation were showed higher Cd content and less effective development than settled biofilms. However diatom communities in younger biofilms exposed to Cd increased their tolerance to Cd by a highly significant development of Nitzschia palea. In contrast, Cd exposure had different effect in installed biofilm and taxonomic composition. These results indicate that mature biofilm may limit Cd accumulation into its architecture and protect diatom communities from the effects of metals.     

Cohesiveness and hydrodynamic properties of young drinking water biofilms

Drinking water biofilms are complex microbial systems mainly composed of clusters of different size and age. Atomic force microscopy (AFM) measurements were performed on 4, 8 and 12 weeks old biofilms in order to quantify the mechanical detachment shear stress of the clusters, to estimate the biofilm entanglement rate ξ. This AFM approach showed that the removal of the clusters occurred generally for mechanical shear stress of about 100 kPa only for clusters volumes greater than 200 μm³. This value appears 1000 times higher than hydrodynamic shear stress technically available meaning that the cleaning of pipe surfaces by water flushing remains always incomplete. To predict hydrodynamic detachment of biofilm clusters, a theoretical model has been developed regarding the averaging of elastic and viscous stresses in the cluster and by including the entanglement rate ξ. The results highlighted a slight increase of the detachment shear stress with age and also the dependence between the posting of clusters and their volume. Indeed, the experimental values of ξ allow predicting biofilm hydrodynamic detachment with same order of magnitude than was what reported in the literature. The apparent discrepancy between the mechanical and the hydrodynamic detachment is mainly due to the fact that AFM mechanical experiments are related to the clusters local properties whereas hydrodynamic measurements reflected the global properties of the whole biofilm.     

Stratification in the cohesion of biofilms grown under various environmental conditions

The objective of this study was to use indirect methods to assess the biofilm cohesion and its vertical stratification. Biofilms were grown under low hydrodynamic strengths in two reactors, a low shear stress reactor (LSSR) or under a defined shear stress in a Couette-Taylor Reactor (CTR), using different electron donors and acceptors. The stratification of biofilm cohesion was characterized from a gradual increase of the hydrodynamic strengths in terms of shear stress (erosion) and abrasion (collisions of particles). Whatever the nature of the biofilms, erosion tests demonstrated a gradual impact of a wall shear stress increase on the biofilms remaining on the substratum surface. These observations demonstrated that the biofilm cohesion was heterogeneous and increased with the biofilm depth. Both erosion and abrasion tests highlighted a basal zone of high cohesion. Investigations based on PCR-SCCP and oxygen uptake rate showed the presence of active microorganisms in this zone.     

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.     

Biofilm responses to ageing and to a high phosphate load in a bench-scale drinking water system

The effects of ageing and of phosphate load on drinking water biofilms developed on a polycarbonate substratum in the pseudo-equilibrium state have been evaluated. Phosphate was added in an amount higher than the stochiometric nutrient requirements of bacteria, at concentrations commonly applied in a drinking water distribution system for corrosion control. Multiple parameters were monitored: heterotrophic plate counts (HPCs), total direct counts (TDCs) and potential exoproteolytic activity (PEPA) in order to characterise changes in bacterial biofilms. The total carbohydrate, amino acid and phosphate contents of biofilms were analysed to characterise and monitor the biochemical composition of the biofilm.The three enumeration methods showed that a pseudo-equilibrium state was reached after 7 weeks of colonisation after which, the bacterial growth rate in the biofilm was 0.1 log per week on average. Bulk phosphate addition doubled the phosphate in the biofilm, but did not affect the other biological, physiological or chemical parameters measured.Polysaccharides increased in the biofilm with ageing and the dynamics of individual carbohydrate synthesis also varied with the age of the biofilm. Once pseudo-equilibrium, it was found that the total proteins were globally constant, whereas the spectra of some individual amino acids of the proteins had significantly changed.     

Impact of microfiltration treatment of secondary wastewater effluent on biofouling of reverse osmosis membranes

The effects of microfiltration (MF) as pretreatment for reverse osmosis (RO) on biofouling of RO membranes were analyzed with secondary wastewater effluents. MF pretreatment reduced permeate flux decline two- to three-fold, while increasing salt rejection. Additionally, the oxygen uptake rate (OUR) in the biofouling layer of the RO membrane was higher for an RO system that received pretreated secondary wastewater effluent compared to a control RO system that received untreated secondary effluent, likely due to the removal of inert particulate/colloidal matter during MF. A higher cell viability in the RO biofilm was observed close to the membrane surface irrespective of pretreatment, which is consistent with the biofilm-enhanced concentration polarization effect. Bacterial 16S rRNA gene clone library analysis revealed dominant biofilm communities of Proteobacteria and Bacteroidetes under all conditions. The Cramer-von Mises test statistic showed that MF pretreatment did not significantly change the bacterial community structure of RO membrane biofilms, though it affected bacterial community structure of non-membrane-associated biofilms (collected from the feed tank wall). The finding that the biofilm community developed on the RO membrane was not influenced by MF pretreatment may imply that RO membranes select for a conserved biofilm community.     

Bacterial activity in plant (Schoenoplectus validus) biofilms of constructed wetlands

Biofilm-bacterial communities have been exploited in the treatment of wastewater in ‘fixed-film’ processes. Our understanding of biofilm dynamics requires a quantitative knowledge of bacterial growth-kinetics in these microenvironments. The aim of this paper was to apply the thymidine assay to quantify bacterial growth without disturbing the biofilm on the surfaces of emergent macrophytes (Schoenoplectus validus) of a constructed wetland. The isotope was rapidly and efficiently taken-up and incorporated into dividing biofilm-bacteria. Isotope diffusion into the biofilm did not limit the growth rate measurement. Isotope dilution was inhibited at >12 μM thymidine. Biofilm-bacterial biomass and growth rates were not correlated to the plant surface area (r² < 0.02). The measurements of in situ biofilm-bacterial growth rates both displayed, and accommodated, the inherent heterogeneity of the complex wetland ecosystem. Biofilm-bacterial respiratory activities, measured using the redox dye CTC, and growth rates were measured simultaneously. The dye did not interfere with bacterial growth. Biofilm-bacterial specific growth rates ranged from 1.4 ± 0.6 d⁻¹ to 3.3 ± 1.3 d⁻¹. In the constructed wetlands of this study biofilm-bacterial specific growth rates, compared to those of natural ecosystems, could be markedly improved through changes in wetland design that increased bacterial respiration while minimising biofilm growth.     

Indoor airborne bacterial communities are influenced by ventilation,occupancy, and outdoor air source

Architects and engineers are beginning to consider a new dimension of indoor air: the structure and composition of airborne microbial communities. A first step in this emerging field is to understand the forces that shape the diversity of bioaerosols across space and time within the built environment. In an effort to elucidate the relative influences of three likely drivers of indoor bioaerosol diversity – variation in outdoor bioaerosols, ventilation strategy, and occupancy load – we conducted an intensive temporal study of indoor airborne bacterial communities in a high‐traffic university building with a hybrid HVAC (mechanically and naturally ventilated) system. Indoor air communities closely tracked outdoor air communities, but human‐associated bacterial genera were more than twice as abundant in indoor air compared with outdoor air. Ventilation had a demonstrated effect on indoor airborne bacterial community composition; changes in outdoor air communities were detected inside following a time lag associated with differing ventilation strategies relevant to modern building design. Our results indicate that both occupancy patterns and ventilation strategies are important for understanding airborne microbial community dynamics in the built environment.     

限制基质条件下生物膜特性的研究

在氮作为限制基质的情况下,采用库爱特-泰勒反应器分别培养异养菌生物膜和异养/自养硝化菌混合生物膜。当生物膜系统达到稳定后,通过提高水力剪切力使生物膜发生脱落,以研究生物膜内层基本特性及活性变化。结果表明,随着水力强度的增加则生物膜发生逐步脱落,脱落前后两种生物膜的特性及微生物活性均发生了较大变化。在两种生物膜内,靠近载膜片的生物膜比靠近液相的生物膜具有更强的粘结力,能抵抗高达10 Pa的水力剪切力,且生物活性较高。对于自养硝化菌生物膜,在生物膜发生脱落后,残余生物膜对氨氮的表面去除速率几乎保持不变,甚至对氨氮的比去除速率还略有增加,说明自养硝化菌可能主要分布在生物膜的内层。     

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.     

Action of a cationic surfactant on the activity and removal of bacterial biofilms formed under different flow regimes

The action of the cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated to control biofilms (aged 7d) formed by Pseudomonas fluorescens on stainless-steel slides, using flow cells reactors, under turbulent and laminar flow. The effect of CTAB was also investigated using planktonic cells in the presence and absence of BSA, by measuring the cellular respiratory activity and the ATP released. The action of CTAB on biofilms was assessed by means of cellular respiratory activity and variation of biofilm mass, immediately and 3, 7 and 12h after the application of CTAB. The physical stability of the biofilm was also assessed using a rotating device, where the effect of the surfactant on the biofilm stability was evaluated through the variation of the mass remaining on the surface. CTAB significantly reduced the activity of the planktonic cells probably due to the rupture of the cells. This effect was significantly reduced in the presence of BSA. Planktonic cells were more easily inactivated than bacteria in biofilms. Biofilms formed under laminar flow were more susceptible than those formed under turbulent flow, but in both cases total inactivation was not achieved. Biofilm recovery was observed, in terms of respiratory activity, in almost all the cases studied. CTAB application by itself did not promote the detachment of biofilms. The physical stability tests showed that the synergistic action of the surfactant and the application of high shear stress to the biofilm increase its detachment.     

Multidimensional modeling of biofilm development and fluid dynamics in a hydrogen-based,membrane biofilm reactor (MBfR)

A two-dimensional, particle-based biofilm model coupled with mass transport and computational fluid dynamics was developed to simulate autotrophic denitrification in a spiral-wound membrane biofilm reactor (MBfR), where hydrogen is supplied via hollow-fiber membrane fabric. The spiral-wound configuration consists of alternating layers of plastic spacer net and membrane fabric that create rows of flow channels, with the top and bottom walls comprised of membranes. The transversal filaments of the spacer partially obstruct the channel flow, producing complex mixing and shear patterns that require multidimensional representation. This study investigated the effect of hydrogen and nitrate concentrations, as well as spacer configuration, on biofilm development and denitrification fluxes. The model results indicate that the cavity spacer filaments, which rest on the bottom membranes, cause uneven biofilm growth. Most biofilm resided on the bottom membranes, only in the wake of the filaments where low shear zones formed. In this way, filament configuration may help achieve a desired biofilm thickness. For the conditions tested in this study, the highest nitrate fluxes were attained by minimizing the filament diameter and maximizing the filament spacing. This lowered the shear stress at the top membranes, allowing for more biofilm growth. For the scenarios studied, biomass limitation at the top membranes hindered performance more significantly than diffusion limitation in the thick biofilms at the bottom membranes. The results also highlighted the importance of two-dimensional modeling to capture uneven biofilm growth on a substratum with geometrical complexity.     

A comparison of bacterial populations in enhanced biological phosphorus removal processes using membrane filtration or gravity sedimentation for solids-liquid separation

In an earlier phase of this study, we compared the performances of pilot scale treatment systems operated in either a conventional enhanced biological phosphorus removal (CEBPR) mode, or a membrane enhanced biological phosphorus removal (MEBPR) mode. In the present investigation, we characterized the bacterial community populations in these processes during parallel operation with the same municipal wastewater feed. The objectives of the study were (1) to assess the similarity of the bacterial communities supported in the two systems over time, (2) to determine if distinct bacterial populations are associated with the MEBPR and CEBPR processes, and (3) to relate the dynamics of the community composition to changes in treatment process configuration and to treatment process performance. The characteristics of the bacterial populations were first investigated with ribosomal intergenic spacer analysis, or RISA. To further understand the bacterial population dynamics, important RISA phylotypes were isolated and identified through 16S RNA gene sequencing.The parallel MEBPR and CEBPR systems developed bacterial communities that were distinct. The CEBPR community appeared to exhibit greater diversity, and this may have been the primary reason why the CEBPR treatment train demonstrated superior functional stability relative to the MEBPR counterpart. Moreover, the more diverse bacterial population apparent in the CEBPR system was observed to be more dynamic than that of the MEBPR process.Several RISA bands were found to be characteristic of either the membrane or conventional biological system. In particular, the MEBPR configuration appeared to be selective for the slow-growing organism Magnospira bakii and for the foam-associated Microthrix parvicella and Gordonia sp., while gravity separation led to the washout of M. parvicella. In both pilot trains, sequence analysis confirmed the presence of EBPR-related organisms such as Accumulibacter phosphatis. The survey of the CEBPR system also revealed many uncultured organisms that have not been well characterized. The study demonstrated that a simple replacement of a secondary clarifier with membrane solids-liquid separation is sufficient to shift the composition of an activated sludge microbial community significantly.     

Effects of fluid-flow velocity and water quality on planktonic and sessile microbial growth in water hydraulic system

Water hydraulic systems use water instead of oil as a pressure medium. Microbial growth in the system may restrict the applicability this technology. The effects of fluid-flow velocity and water quality on microbial growth and biofilm formation were studied with a pilot-scale water hydraulic system. The fluid-flow velocities were 1.5-5.2 m/s and the corresponding shear stresses 9.1-84 N/m2. The fluid-flow velocity had an insignificant effect on the total bacterial numbers and the numbers of viable heterotrophic bacteria in the pressure medium. Microbial attachment occurred under high shear stresses. The fluid-flow velocity did not affect the biofilm formation in the tank. Increase in the flow velocity decreased the bacterial densities on the pipe surfaces indicating preferable biofilm formation on areas with low flow velocity. Using ultrapure water as the pressure medium decreased the total cell numbers and resulted in slower growth of bacteria in the pressure medium. Lowering the nutrient concentration retarded biofilm formation but did not affect the final cell densities. The decreasing pressure medium nutrient concentration favoured microbial attachment in the tank instead of the pipelines. In conclusion, microbial growth and biofilm formation in water hydraulic systems cannot be controlled by the fluid-flow velocity or the quality of the pressure medium.     

Comparative microscale analysis of the effects of triclosan and triclocarban on the structure and function of river biofilm communities

The broad spectrum antimicrobials triclosan (TCS) and triclocarban (TCC) are commonly detected in the environment. However, there is very limited understanding of the aquatic ecological implications of these agents. During this study, river biofilms were cultivated using 10 µg l^− 1 of TCS or TCC and the equivalent in nutrients (carbon, nitrogen) over a developmental period of 8 weeks. Confocal laser microscopy showed that the biofilm communities developing under the influence of TCS and TCC had community architecture and composition different from either control or nutrient exposed communities. Microscale analyses of biofilm community structure indicated a significant reduction in algal biomass (p < 0.05) as a result of exposure to either TCS or TCC. Thymidine incorporation did not detect significant differences between control and treated communities. The use of carbon utilization assays based on growth indicated that, in general, TCS and TCC suppressed utilization. The community was altered from one dominated by autotrophic processes to one dominated by heterotrophic processes. Both TCS and TCC treatments resulted in significant (p < 0.05) alterations in the composition of the EPS matrix of the communities, suggesting significant changes in community composition. Denaturing gradient gel electrophoresis and PCA-ANOSIM analyses indicated a significant change occurred in the bacterial community as a consequence of TCS treatments. Enumeration of micrometazoa and protozoa revealed an increase in micrometazoan numbers over control values, whereas no clear impact on protozoa was detected in any treatment. This study indicated significant effects of 10 µg l^− 1 TCS and TCC on microbial community composition, algal biomass, architecture and activity.     

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

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

Bacterial community composition and structure of biofilms developing on nanofiltration membranes applied to wastewater treatment

The structure and microbial communities of biofilms developing on cross-flow nanofiltration (NF) membranes at different temperatures (20, 25 or 34 degrees C) and operation lengths (8h-24days) were studied. Feedwater comprised tertiary quality wastewater effluent or synthetic media mimicking effluents of intermediate quality. After each run, the membranes were autopsied for bacterial enumeration, bacterial community composition and microscopy visualization (SEM, CLSM and AFM/NSOM). Community composition was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) coupled with sequence analysis of 16S rRNA gene fragments from dominant bands. Deposition of polysaccharides and initial bacterial colonization were observed within 8h, whereas developed biofilms markedly affecting membrane permeability were evident from days 2-3 onwards. Regardless of applied conditions, the heterotrophic plate counts in the biofilm were 3-4x10(6)CFU/cm(2) and the thickness of the biofouling layer was 20-30microm. From a total of 22 sequences obtained from 14 independent experiments, most species identified were Gram negative (19 of 22 sequences). Proteobacteria were found to be a prevalent group in all cases (16 of 22 sequences) and among it, the beta-subclass was the most predominant (8 sequences), followed by the gamma-subclass (5 sequences). Pseudomonas/Burkholderia, Ralstonia, Bacteroidetes and Sphingomonas were the dominant groups found in most cases. Even though the microbial population might be important with respect to biofouling patterns, membrane permeability decline seems to be more substantially influenced by the formation and accumulation of exopolymeric substances (EPS).