Modelling biofilm development: The importance of considering the link between EPS distribution, detachment mechanisms and physical properties.

In industry, treatments against biofilms need to be optimized and, in the wastewater treatment field, biofilm composition needs to be controlled. Therefore, describing the biochemical and physical structures of biofilms is now required to better understand the influence of operating parameters and treatment on biofilms. The present study aims to investigate how growth conditions influence EPS composition, biofilm physical properties and volume detachment using a 1D biofilm model. Two types of EPS are considered in the present model, proteins and polysaccharides. The main hypotheses are that: (i) the production of polysaccharides occurs mainly under strong nutrient limitation(s) while the production of proteins is coupled to both the substrate uptake rate and the lysis process; (ii) the local biofilm porosity depends on the local biofilm composition. Both volume and surface detachment occur in biofilms and volume detachment extent depends on the biofilm local cohesion and thus on the local composition of biofilms for a given shear stress. The model is based on experimental trends and aims to represent these observations on the basis of biochemical and physical processes. Four case studies covering a wide range of contrasting growth conditions such as different COD/N ratios, applied SOLR and shear stresses are investigated. The model predicts how the biochemical and physical biofilm structures change as a result of contrasting growth conditions. More precisely simulation results are in good agreement with the main experimental observations reported in the literature, such as: (i) a strong nitrogen limitation of growth induces an important accumulation of polysaccharides leading to a more porous and homogenous biofilm, (ii) a high applied surface organic loading load allows to obtain a high biofilm thickness, (iii) a strong shear stress applied during the biofilm growth leads to a reduction of the biofilm thickness and to a consolidation of the biofilm structure. Overall, this model represents a relevant decision tool for the selection of appropriate enzymatic treatments in the context of negative biofilm control. From our results, it appears that protease based treatments should be more appropriate for biofilms developed under low COD/N ratios (about 20 gCOD/gN) whereas both glucosidases and proteases based treatments should be more appropriate for biofilms developed under high COD/N ratio (about 70 gCOD/gN). In addition, the model could be useful for other applications such as resource recovery in biofilms or granules, and help to better understand biological membrane fouling.

Particulate substrate retention in plug-flow and fully-mixed conditions during operation of aerobic granular sludge systems.

Particulate substrate (XB) is the major organic substrate fraction in most municipal wastewaters. However, the impact of XB on aerobic granular sludge (AGS) systems is not fully understood. This study evaluated the physical retention of XB in AGS sequencing batch reactor (SBR) during anaerobic plug-flow and then aerobic fully-mixed conditions. The influence of different sludge types and operational variables on the extent and mechanisms of XB retention in AGS SBR were evaluated. XB mass-balancing and magnetic resonance imaging (MRI) were applied. During the anaerobic plug-flow feeding, most XB was retained in the first few cm of the settled sludge bed within the interstitial voids, where XB settled and accumulated ultimately resulting in the formation of a filter-cake. Sedimentation and surface filtration were thus the dominant XB retention mechanisms during plug-flow conditions, indicating that contact and attachment of XB to the biomass was limited. XB retention was variable and influenced by the XB influent concentration, sludge bed composition and upflow feeding velocity (vww). XB retention increased with larger XB influent concentrations and lower vww, which demonstrated the importance of sedimentation on XB retention during plug-flow conditions. Hence, large fractions of influent XB likely re-suspended during aerobic fully-mixed conditions, where XB then preferentially and rapidly attached to the flocs. During fully-mixed conditions, increasing floc fractions, longer mixing times and larger XB concentrations increased XB retention. Elevated XB retention was observed after short mixing times < 60 min when flocs were present, and the contribution of flocs towards XB retention was even more pronounced for short mixing times < 5 min. Overall, our results suggest that flocs occupy an environmental niche that results from the availability of XB during aerobic fully-mixed conditions of AGS SBR. Therefore, a complete wash-out of flocs is not desirable in AGS systems treating municipal wastewater.

Organic substrate diffusibility governs microbial community composition, nutrient removal performance and kinetics of granulation of aerobic granular sludge.

Basic understanding of formation of aerobic granular sludge (AGS) has mainly been derived from lab-scale systems with simple influents containing only highly diffusible volatile fatty acids (VFA) as organic substrate. This study compares start-up of AGS systems fed by different synthetic and municipal wastewaters (WW), characterised by increasing complexity in terms of non-diffusible organic substrate. Four AGS reactors were started with the same inoculum activated sludge and operated for one year. The development of AGS, settling characteristics, nutrient and substrate removal performance as well as microbial community composition were monitored. Our results indicate that the higher the content of diffusible organic substrate in the WW, the faster the formation of AGS. The presence of non-diffusible organic substrate in the influent WW led to the formation of small granules and to the presence of 20-40% (% of total suspended solids) of flocs in the AGS. When AGS was fed with complex influent WW, the classical phosphorus and glycogen accumulating organisms (PAO, GAO) were outcompeted by their fermentative equivalents. Substrate and nutrient removal was observed in all reactors, despite the difference in physical and settling properties of the AGS, but the levels of P and N removal depended on the influent carbon composition. Mechanistically, our results indicate that increased levels of non-diffusible organic substrate in the influent lower the potential for microbial growth deep inside the granules. Additionally, non-diffusible organic substrates give a competitive advantage to the main opponents of AGS formation - ordinary heterotrophic organisms (OHO). Both of these mechanisms are suspected to limit AGS formation. The presented study has relevant implications for both practice and research. Start-up duration of AGS systems treating high complexity WW were one order of magnitude higher than a typical lab-scale system treating VFA-rich synthetic WW, and biomass as flocs persisted as a significant fraction. Finally, the complex synthetic influent WW - composed of VFA, soluble fermentable and particulate substrate - tested here seems to be a more adequate surrogate of real municipal WW for laboratory studies than 100%-VFA WW.

Pore-Scale Hydrodynamics in a Progressively Bioclogged Three-Dimensional Porous Medium: 3-D Particle Tracking Experiments and Stochastic Transport Modeling.

Biofilms are ubiquitous bacterial communities that grow in various porous media including soils, trickling, and sand filters. In these environments, they play a central role in services ranging from degradation of pollutants to water purification. Biofilms dynamically change the pore structure of the medium through selective clogging of pores, a process known as bioclogging. This affects how solutes are transported and spread through the porous matrix, but the temporal changes to transport behavior during bioclogging are not well understood. To address this uncertainty, we experimentally study the hydrodynamic changes of a transparent 3-D porous medium as it experiences progressive bioclogging. Statistical analyses of the system's hydrodynamics at four time points of bioclogging (0, 24, 36, and 48 h in the exponential growth phase) reveal exponential increases in both average and variance of the flow velocity, as well as its correlation length. Measurements for spreading, as mean-squared displacements, are found to be non-Fickian and more intensely superdiffusive with progressive bioclogging, indicating the formation of preferential flow pathways and stagnation zones. A gamma distribution describes well the Lagrangian velocity distributions and provides parameters that quantify changes to the flow, which evolves from a parallel pore arrangement under unclogged conditions, toward a more serial arrangement with increasing clogging. Exponentially evolving hydrodynamic metrics agree with an exponential bacterial growth phase and are used to parameterize a correlated continuous time random walk model with a stochastic velocity relaxation. The model accurately reproduces transport observations and can be used to resolve transport behavior at intermediate time points within the exponential growth phase considered.

Biofilm increases permeate quality by organic carbon degradation in low pressure ultrafiltration.

We investigated the influence of biofouling of ultrafiltration membranes on the removal of organic model foulants and ultimately on the quality of permeate. Gravity Driven Membrane ultrafiltration (GDM) membrane systems were operated with modified river water during five weeks without control of the biofilm formation. Three GDM systems were studied: two systems with biofilms exposed to (A) variable or (B) constant load of organic foulants, and (C) one system operated without biofilm and exposed to constant foulant loading. Biodegradable dextran or non-biodegradable polystyrene sulfonate model foulants were tested. Substrate biodegradability was confirmed by Size Exclusion Chromatography (SEC) and by degradation batch tests (D). The GDM systems (A) and (B) were fed with pre-filtered river water supplemented with dextran (Dex) of 1, 150 or 2000 kDa, or polystyrene sulfonate (PSS) of 1 or 80 kDa at concentrations of 2-3.5 mgC L(-1). In exp. (C) the feed water consisted of deionized water with 25 mgC L(-1) of either PSS 1, 80 kDa or Dex 2000 kDa. The biofilm formation on UF membrane surfaces controlled the foulant permeation and thus the permeate quality. Biofilms exposed to continuous foulant loading (exp. B) degraded low molecular weight (LMW) biodegradable foulants (1 kDa Dex), which improved the permeate quality. For high molecular weight (HMW) substrates (150, 2000 kDa Dex), the improvement of the permeate quality was observed after 7 days of biofilm formation, and resulted from the foulant hydrolysis followed by degradation. For non-biodegradable foulants, an improvement of 20% of the retention was observed for the polystyrene (1, 80 kDa PSS) due to the presence of biofilms on membrane surfaces. For variable foulant loading (exp. A) the biofilms hydrolysed the large biodegradable foulants but did not degraded them fully, which resulted a deterioration of the permeate quality (except for the LMW dextran (1 kDa) that was fully degraded). Overall, the "biofilm + membrane" composite retained a larger amount of biodegradable foulant than the membrane alone, due to the activity of the biofilm. However, this resulted in an increased biofilm accumulation and reduced flux. In presence of the biofilm, the highest fluxes were observed for control (no foulant) and for small non-biodegradable foulants (PSS 1 kDa). Low fluxes were observed for the accumulating on membrane surface or degradable foulants (exp. B). But, the lowest fluxes were observed in absence of the biofilm (exp. C) due to physical accumulation of the foulants (PSS 80 kDa and Dextran 2000 kDa). Overall our study demonstrates that the presence of biofilms on membrane surfaces has some benefits: (i) biofilm helps to increase the permeate quality and (ii) biofilms protect the membrane from further fouling. Permeate flux stabilizes in the case of biofilm-membrane composite, while it continuously declines in the case of the membrane only.

Inorganic particles increase biofilm heterogeneity and enhance permeate flux.

This study investigated the influence of inorganic particles on the hydraulic resistance of biofilm grown on membrane surface during low-pressure dead-end ultrafiltration. Gravity-driven ultrafiltration membrane systems were operated during several weeks without any flushing or cleaning. Smaller (kaolin d0.5 = 3.6 µm) or larger (kaolin with diatomaceous earth 50/50%, d0.5 = 18.1 µm) particles were added to pre-filtered creek water or to unfiltered creek water. It was demonstrated in both experiments that presence of finer particles in the feed water (kaolin) induced formation of compact and homogeneous biofilm structure. On the other hand presence of the larger particles (diatomite) helped to counterbalance the effect of fine particles due to the formation of more heterogeneous and permeable biofilm structure. The hydraulic resistance of biofilms formed with fine particles was significantly higher than the resistance of biofilm formed in (1) absence of any inorganic particles or (2) in presence of the mixed particle population. The membrane orientation (vertical or horizontal) determined which particles were accumulating at the membrane surface, with structural differences shown by Scanning Electron Microscopy (SEM). For vertical membranes, the larger particles were selectively removed due to sedimentation and did not contribute to the biofilm development. Thus the selection of smaller particles due to vertical membrane configuration negatively affected the biofilm structure and permeation rates, and such selective accumulation of fine particles should be avoided.

Distribution and hydrophobic properties of Extracellular Polymeric Substances in biofilms in relation towards cohesion.

A heterotrophic biofilm (B1) and a mixed autotrophic-heterotrophic biofilm (B2) were developed in an annular reactor and submitted to an erosion test in order to selectively detach top layers from the bottom layers. Densities of the basal layers were 5-fold higher and 3-fold higher than the densities of the entire biofilms B1 and B2, respectively. After extraction, EPS content in B1 biofilm was found higher in the basal layer (95 mg g⁻¹ VSS) compared to the top layer (30 mg g⁻¹ VSS), while B2 biofilm had a higher EPS content in the top layer (303 mg g⁻¹ VSS) compared to the basal layer (135 mg g⁻¹ VSS). Hydrophobic Interaction Chromatography (HIC) indicates that hydrophobic EPS (HEPS) in both biofilms reached 21% of EPS in basal cohesive layers, and remained slightly lower or identical (16-19%) in top detached biofilm layers. Strong interacting HEPS were found in a higher proportion in the mixed autotrophic-heterotrophic B2 which was also more diversified in terms of bacterial populations than the B1 heterotrophic biofilm. These results show that HEPS content correlates better with cohesive properties of the biofilm layers than global EPS content and that strong hydrophobic adhesion forces may be related to microbial populations such as the presence of nitrifiers.

Cohesion and detachment in biofilm systems for different electron acceptor and donors.

This work deals with the cohesion and detachment in biofilm systems for two electron acceptors and for two electron donors. Biofilms were developed on plates, under very low shear stress for one month and then subjected to an erosion test for two hours in a Couette-Taylor reactor. Biofilm was characterised in terms of average thickness and residual TOC mass. It was found that the biofilm structure is very heterogeneous and stratified. The top layer, which represents 60% of the biofilm mass, is very fragile and can be easily detached; the basal layer, which represents 20% of the biofilm mass, is very cohesive and can resist shear stresses up to 13 Pa. Between these two layers, a middle layer of intermediary cohesion represents 20% of the initial biofilm mass.