A novel approach to investigate biofilm accumulation and bacterial transport in porous matrices

Knowledge of bacterial transport through, and biofilm growth in, porous media is vitally important in numerous natural and engineered environments. Despite this, porous media systems are generally oversimplified and the local complexity of cell transport, biofilm formation and the effect of biofilm accumulation on flow patterns is lost. In this study, cells of the sulphate-reducing bacterium, Desulfovibrio sp. EX265, accumulated primarily on the leading faces of obstructions and developed into biofilm, which grew to narrow and block pore throats (at a rate of 12 micro m h(-1) in one instance). This pore blocking corresponded to a decrease in permeability from 9.9 to 4.9 Darcy. Biofilm processes were observed in detail and quantitative data were used to describe the rate of biofilm accumulation temporally and spatially. Accumulation in the inlet zone of the micromodel was 10% higher than in the outlet zone and a mean biofilm height of 28.4 micro m was measured in a micromodel with an average pore height of 34.9 microm. Backflow (flow reversal) of fluid was implemented on micromodels blocked with biofilm growth. Although biofilm surface area cover did immediately decrease (approximately 5%), the biofilm quickly re-established and permeability was not significantly affected (9.4 Darcy). These results demonstrate that the glass micromodel used here is an effective tool for in situ analysis and quantification of bacteria in porous media.     

Pore-scale investigation of biomass plug development and propagation in porous media.

Biomass plugging of porous media finds application in enhanced oil recovery and bioremediation. An understanding of biomass plugging of porous media was sought by using a porous glass micromodel through which biomass and nutrient were passed. This study describes the pore-scale physics of biomass plug propagation of Leuconostoc mesenteroides under nutrient-rich conditions. It was found that, as the nutrient flowed through the micromodel, the initial biomass plug occurred at the nutrient-inoculum interface due to growth in the larger pore throats. As growth proceeded, biomass filled and closed these larger pore throats, until only isolated groupings of pore throats with smaller radii remained empty. As nutrient flow continued, a maximum pressure drop was reached. At the maximum pressure drop, the biomass yielded in a manner similar to a Bingham plastic to form a breakthrough channel consisting of a path of interconnected pore throats. The channel incorporated the isolated groupings of empty pore throats that had been present before breakthrough. As the nutrient flow continued, subsequent plugs developed as breakthrough channels refilled with biomass and in situ growth was stimulated in the region just downstream of the previous plug. The downstream plugs had a higher fraction of isolated groupings of empty pore throats, which can be attributed to depletion of nutrient downstream. When the next breakthrough channel formed, it incorporated these isolated groupings, causing the breakthrough channels to be branched. It was observed that the newly formed plug could be less stable with this higher fraction of empty pore throats and that the location of breakthrough channels changed in subsequent plugs. This change in breakthrough channel location could be attributed to the redistribution of nutrient flow and the changes in flowrate in the pore throats.     

Biofilm development and the dynamics of preferential flow paths in porous media

A two-dimensional pore-scale numerical model was developed to evaluate the dynamics of preferential flow paths in porous media caused by bioclogging. The liquid flow and solute transport through the pore network were coupled with a biofilm model including biomass attachment, growth, decay, lysis, and detachment. Blocking of all but one flow path was obtained under constant liquid inlet flow rate and biomass detachment caused by shear forces only. The stable flow path formed when biofilm detachment balances growth, even with biomass weakened by decay. However, shear forces combined with biomass lysis upon starvation could produce an intermittently shifting location of flow channels. Dynamic flow pathways may also occur when combined liquid shear and pressure forces act on the biofilm. In spite of repeated clogging and unclogging of interconnected pore spaces, the average permeability reached a quasi-constant value. Oscillations in the medium permeability were more pronounced for weaker biofilms.     

A three-dimensional computer model analysis of three hypothetical biofilm detachment mechanisms

Three hypothetical mechanisms of detachment were incorporated into a three-dimensional computer model of biofilm development. The model integrated processes of substrate utilization, substrate diffusion, growth, cell advection, and detachment in a cellular automata framework. The purpose of this investigation was to characterize each of the mechanisms with respect to four criteria: the resulting biofilm structure, the existence of a steady state, the propensity for sloughing events, and the dynamics during starvation. The three detachment mechanisms analyzed represented various physical and biological influences hypothesized to affect biofilm detachment. The first invoked the concept of fluid shear removing biomass that protrudes far above the surface and is therefore subjected to relatively large drag forces. The second pathway linked detachment to changes in the local availability of a nutrient. The third pathway simulated an erosive process in which individual cells are lost from the surface of a biofilm cell cluster. The detachment mechanisms demonstrated diverse behaviors with respect to the four analysis criteria. The height-dependant mechanism produced flat, steady state biofilms that lacked sloughing events. Detachment based on substrate limitation produced significant sloughing events. The resulting biofilm structures included distinct, hollow clusters separated by channels. The erosion mechanism produced neither a non-zero steady state nor sloughing events. A mechanism combining all three-detachment mechanisms produced mushroom-like structures. The dynamics of biofilm decay during starvation were distinct for each detachment mechanism. These results show that detachment is a critical determinant of biofilm structure and of the dynamics of biofilm accumulation and loss.     

Pore‐network modeling of biofilm evolution in porous media

The influence of bacterial biomass on hydraulic properties of porous media (bioclogging) has been explored as a viable means for optimizing subsurface bioremediation and microbial enhanced oil recovery. In this study, we present a pore network simulator for modeling biofilm evolution in porous media including hydrodynamics and nutrient transport based on coupling of advection transport with Fickian diffusion and a reaction term to account for nutrient consumption. Biofilm has non‐zero permeability permitting liquid flow and transport through the biofilm itself. To handle simultaneous mass transfer in both liquid and biofilm in a pore element, a dual‐diffusion mass transfer model is introduced. The influence of nutrient limitation on predicted results is explored. Nutrient concentration in the network is affected by diffusion coefficient for nutrient transfer across biofilm (compared to water/water diffusion coefficient) under advection dominated transport, represented by mass transport Péclet number >1. The model correctly predicts a dependence of rate of biomass accumulation on inlet concentration. Poor network connectivity shows a significantly large reduction of permeability, for a small biomass pore volume. Biotechnol. Bioeng. 2011;108: 2413–2423. © 2011 Wiley Periodicals, Inc.     

Hypothesis for the role of nutrient starvation in biofilm detachment

A combination of experimental and theoretical approaches was used to investigate the role of nutrient starvation as a potential trigger for biofilm detachment. Experimental observations of detachment in a variety of biofilm systems were made with pure cultures of Pseudomonas aeruginosa. These observations indicated that biofilms grown under continuous-flow conditions detached after flow was stopped, that hollow cell clusters were sometimes observed in biofilms grown in flow cells, and that lysed cells were apparent in the internal strata of colony biofilms. When biofilms were nutrient starved under continuous-flow conditions, detachment still occurred, suggesting that starvation and not the accumulation of a metabolic product was responsible for triggering detachment in this particular system. A cellular automata computer model of biofilm dynamics was used to explore the starvation-dependent detachment mechanism. The model predicted biofilm structures and dynamics that were qualitatively similar to those observed experimentally. The predicted features included centrally located voids appearing in sufficiently large cell clusters, gradients in growth rate within these clusters, and the release of most of the biofilm with simulated stopped-flow conditions. The model was also able to predict biofilm sloughing resulting solely from this detachment mechanism. These results support the conjecture that nutrient starvation is an environmental cue for the release of microbes from a biofilm.     

Hypothesis for the Role of Nutrient Starvation in Biofilm Detachment

A combination of experimental and theoretical approaches was used to investigate the role of nutrient starvation as a potential trigger for biofilm detachment. Experimental observations of detachment in a variety of biofilm systems were made with pure cultures of Pseudomonas aeruginosa. These observations indicated that biofilms grown under continuous-flow conditions detached after flow was stopped, that hollow cell clusters were sometimes observed in biofilms grown in flow cells, and that lysed cells were apparent in the internal strata of colony biofilms. When biofilms were nutrient starved under continuous-flow conditions, detachment still occurred, suggesting that starvation and not the accumulation of a metabolic product was responsible for triggering detachment in this particular system. A cellular automata computer model of biofilm dynamics was used to explore the starvation-dependent detachment mechanism. The model predicted biofilm structures and dynamics that were qualitatively similar to those observed experimentally. The predicted features included centrally located voids appearing in sufficiently large cell clusters, gradients in growth rate within these clusters, and the release of most of the biofilm with simulated stopped-flow conditions. The model was also able to predict biofilm sloughing resulting solely from this detachment mechanism. These results support the conjecture that nutrient starvation is an environmental cue for the release of microbes from a biofilm.     

Two-dimensional model of biofilm detachment caused by internal stress from liquid flow

A two-dimensional model for biofilm growth and detachment was used to evaluate the effect of detachment on biofilm structures. The detachment process is considered to be due to internal stress created by moving liquid past the biofilm. This model generated a variety of realistic biofilm-formation patterns. It was possible to model in a unified way two different biofilm detachment processes, erosion (small-particle loss), and sloughing (large-biomass-particle removal). The distribution of the fraction from total biomass detached as a function of detached particle mass, gives indications about which of the two mechanisms is dominant. Model simulations indicate that erosion makes the biofilm surface smoother. Sloughing, in contrast, leads to an increased biofilm-surface roughness. Faster growing biofilms have a faster detachment rate than slow-growing biofilms, under similar hydrodynamic conditions and biofilm strength. This is in perfect accordance with the experimental evidence showing that detachment is dependent on both shear- and microbial-growth rates. High growth rates trigger instability in biofilm accumulation and abrupt biomass loss (sloughing). Massive sloughing can be avoided by high liquid shear, combined with low biomass growth rates. As the modeling results show, the causes for sloughing must be sought not only in the biofilm strength, but also in its shape. Several "mushroom-like" biofilm structures like those repeatedly reported in the literature occurred, due to a combined effect of nutrient depletion and breaking at the colony base. A rough carrier surface promotes biofilm development in hydrodynamic conditions in which the biofilm on a flat surface would not form. Although biofilm patches filled completely the cavity in which they started to grow, they were unable to spill over the carrier peaks and to fully colonize the substratum.     

Biomass plug development and propagation in porous media

Exopolymer-producing bacteria can be used to modify soil profiles for enhanced oil recovery or bioremediation. Understanding the mechanisms associated with biomass plug development and propagation is needed for successful application of this technology. These mechanisms were determined from packed-bed and micromodel experiments that simulate plugging in porous media. Leuconostoc mesenteroides was used, because production of dextran, a water-insoluble exopolymer, can be controlled by using different carbon sources. As dextran was produced, the pressure drop across the porous media increased and began to oscillate. Three pressure phases were identified under exopolymer-producing conditions: the exopolymer-induction phase, the plugging phase, and the plug-propagation phase. The exopolymer-induction phase extended from the time that exopolymer-producing conditions were induced until there was a measurable increase in pressure drop across the porous media. The plugging phase extended from the first increase in pressure drop until a maximum pressure drop was reached. Changes in pressure drop in these two phases were directly related to biomass distribution. Specifically, flow channels within the porous media filled with biomass creating a plugged region where convective flow occurred only in water channels within the biofilm. These water channels were more restrictive to flow causing the pressure drop to increase. At a maximum pressure drop across the porous media, the biomass yielded much like a Bingham plastic, and a flow channel was formed. This behavior marked the onset of the plug-propagation phase which was characterized by sequential development and breakthrough of biomass plugs. This development and breakthrough propagated the biomass plug in the direction of nutrient flow. The dominant mechanism associated with all three phases of plugging in porous media was exopolymer production; yield stress is an additional mechanism in the plug-propagation phase.     

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

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

Flowing biofilms as a transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor system

Fluid flow has been shown to be important in influencing biofilm morphology and causing biofilms to flow over surfaces in flow cell experiments. However, it is not known whether similar effects may occur in porous media. Generally, it is assumed that the primary transport mechanism for biomass in porous media is through convection, as suspended particulates (cells and flocs) carried by fluid flowing through the interstices. However, the flow of biofilms over the surfaces of soils and sediment particles, may represent an important flux of biomass, and subsequently affect both biological activity and permeability. Mixed species bacterial biofilms were grown in glass flow cells packed with 1 mm diameter glass beads, under laminar or turbulent flow (porous media Reynolds number = 20 and 200 respectively). The morphology and dynamic behavior reflected those of biofilms grown in the open flow cells. The laminar biofilm was relatively uniform and after 23 d had inundated the majority of the pore spaces. Under turbulent flow the biofilm accumulated primarily in protected regions at contact points between the beads and formed streamers that trailed from the leeward face. Both biofilms caused a 2 to 3-fold increase in friction factor and in both cases there were sudden reductions in friction factor followed by rapid recovery, suggesting periodic sloughing and regrowth events. Time-lapse microscopy revealed that under both laminar and turbulent conditions biofilms flowed over the surface of the porous media. In some instances ripple structures formed. The velocity of biofilm flow was on the order of 10 mum h(-1) in the turbulent flow cell and 1.0 mum h(-1) in the laminar flow cell.     

The effect of biofilm permeability on bio-clogging of porous media

A 3D Biofilm model, appropriate for complex porous media support structures, is successfully modified such that non‐zero permeability of biofilms structures is enabled. A systematic study is then conducted into the influence of biofilm permeability on overall biomass growth rate. This reveals a significant influence at large biofilm concentrations; even when the permeability of the biomass is 1.25% of that of the free pore space, biomass accumulation increased by a factor of ～3 over 40 h. The effect is shown to be retained when allowing for biomass detachment or erosion as a consequence of adjacent velocity shear. We conclude that biofilm permeability should be included in biofilm models and that further experimental work is required to better describe the link between biofilm permeability and local microstructure. Biotechnol. Bioeng. 2012; 109:1031–1042. © 2011 Wiley Periodicals, Inc.     

Towards optimum permeability reduction in porous media using biofilm growth simulations

While biological clogging of porous systems can be problematic in numerous processes (e.g., microbial enhanced oil recovery—MEOR), it is targeted during bio‐barrier formation to control sub‐surface pollution plumes in ground water. In this simulation study, constant pressure drop (CPD) and constant volumetric flow rate (CVF) operational modes for nutrient provision for biofilm growth in a porous system are considered with respect to optimum (minimum energy requirement for nutrient provision) permeability reduction for bio‐barrier applications. Biofilm growth is simulated using a Lattice‐Boltzmann (LB) simulation platform complemented with an individual‐based biofilm model (IbM). A biomass detachment technique has been included using a fast marching level set (FMLS) method that models the propagation of the biofilm–liquid interface with a speed proportional to the adjacent velocity shear field. The porous medium permeability reduction is simulated for both operational modes using a range of biofilm strengths. For stronger biofilms, less biomass deposition and energy input are required to reduce the system permeability during CPD operation, whereas CVF is more efficient at reducing the permeability of systems containing weaker biofilms. Biotechnol. Bioeng. 2009;103: 767–779. © 2009 Wiley Periodicals, Inc.     

Biofilm formation and sloughing in Serratia marcescens are controlled by quorum sensing and nutrient cues

We describe here a role for quorum sensing in the detachment, or sloughing, of Serratia marcescens filamentous biofilms, and we show that nutrient conditions affect the biofilm morphotype. Under reduced carbon or nitrogen conditions, S. marcescens formed a classical biofilm consisting of microcolonies. The filamentous biofilm could be converted to a microcolony-type biofilm by switching the medium after establishment of the biofilm. Similarly, when initially grown as a microcolony biofilm, S. marcescens could be converted back to a filamentous biofilm by increasing the nutrient composition. Under high-nutrient conditions, an N-acyl homoserine lactone quorum-sensing mutant formed biofilms that were indistinguishable from the wild-type biofilms. Similarly, other quorum-sensing-dependent behaviors, such as swarming motility, could be rendered quorum sensing independent by manipulating the growth medium. Quorum sensing was also found to be involved in the sloughing of the filamentous biofilm. The biofilm formed by the bacterium consistently sloughed from the substratum after approximately 75 to 80 h of development. The quorum-sensing mutant, when supplemented with exogenous signal, formed a wild-type filamentous biofilm and sloughed at the same time as the wild type, and this was independent of surfactant production. When we removed the signal from the quorum-sensing mutant prior to the time of sloughing, the biofilm did not undergo significant detachment. Together, the data suggest that biofilm formation by S. marcescens is a dynamic process that is controlled by both nutrient cues and the quorum-sensing system.     

A new approach to model the spatiotemporal development of biofilm phase in porous media

Bacteria can exist within biofilms that are attached to the solid matrix of a porous medium. Under certain conditions, the biomass can fully occupy the pore space leading to reduced hydraulic conductivity and mass transport. Here, by treating biofilm as a growing, high-viscosity phase, a novel macroscopic approach to model biofilm spatial expansion and its corresponding effects on porous medium hydraulic properties is presented. The separate yet coupled flow of the water and biofilm phases is handled by using relative permeability curves that allow for biofilm movement within the porous medium and bioclogging effects. Fluid flow is governed by Darcy's law and component transport is set by the convection-diffusion equation reaction terms for each component. Here, the system of governing equations is solved by using a commercial multiphase flow reservoir simulator, which is used to validate the model against published laboratory experiments. A comparison of the model and experimental observations reveal that the model provides a reasonable means to predict biomass development in the porous medium. The results reveal that coupled flow of water and movement of biofilm, as described by relative permeability curves, is complex and has a large impact on the development of biomass and consequent bioclogging in the porous medium.     

Anomalous spatial redistribution of competing bacteria under starvation conditions

Bacterial cells evolved under prolonged stress often have a growth advantage in stationary phase (GASP); we expect GASP cells to maintain a proliferative state and dominate wild-type cells during starvation, especially when nutrients are limited and the medium has been conditioned. However, when we compete GASP mutants against wild-type cells in a chain of microfluidic microhabitat patches (MHPs) with alternating nutrient-rich and nutrient-limited regions, we observe the reverse effect: wild-type cells achieve maximum relative density under nutrient-limited conditions, while GASP cells dominate nutrient-rich regions. We explain this surprising observation in terms of ideal free distributions, where we show that wild-type cells maximize their fitness at high cell density by redistributing themselves to sparsely populated MHPs. At the microscopic level, we describe how biofilm formation also contributes to the population redistribution. We conclude by discussing the implications of these results for social interactions of more complex organisms.     

Flow cell hydrodynamics and their effects on E. coli biofilm formation under different nutrient conditions and turbulent flow

Biofilm formation is a major factor in the growth and spread of both desirable and undesirable bacteria as well as in fouling and corrosion. In order to simulate biofilm formation in industrial settings a flow cell system coupled to a recirculating tank was used to study the effect of a high (550 mg glucose l?1) and a low (150 mg glucose l?1) nutrient concentration on the relative growth of planktonic and attached biofilm cells of Escherichia coli JM109(DE3). Biofilms were obtained under turbulent flow (a Reynolds number of 6000) and the hydrodynamic conditions of the flow cell were simulated by using computational fluid dynamics. Under these conditions, the flow cell was subjected to wall shear stresses of 0.6 Pa and an average flow velocity of 0.4 m s?1 was reached. The system was validated by studying flow development on the flow cell and the applicability of chemostat model assumptions. Full development of the flow was assessed by analysis of velocity profiles and by monitoring the maximum and average wall shear stresses. The validity of the chemostat model assumptions was performed through residence time analysis and identification of biofilm forming areas. These latter results were obtained through wall shear stress analysis of the system and also by assessment of the free energy of interaction between E. coli and the surfaces. The results show that when the system was fed with a high nutrient concentration, planktonic cell growth was favored. Additionally, the results confirm that biofilms adapt their architecture in order to cope with the hydrodynamic conditions and nutrient availability. These results suggest that until a certain thickness was reached nutrient availability dictated biofilm architecture but when that critical thickness was exceeded mechanical resistance to shear stress (ie biofilm cohesion) became more important.     

Flowing biofilms as a transport mechanism for biomass through porous media under laminar and turbulent conditions in a laboratory reactor system

Abstract

Fluid flow has been shown to be important in influencing biofilm morphology and causing biofilms to flow over surfaces in flow cell experiments. However, it is not known whether similar effects may occur in porous media. Generally, it is assumed that the primary transport mechanism for biomass in porous media is through convection, as suspended particulates (cells and flocs) carried by fluid flowing through the interstices. However, the flow of biofilms over the surfaces of soils and sediment particles, may represent an important flux of biomass, and subsequently affect both biological activity and permeability. Mixed species bacterial biofilms were grown in glass flow cells packed with 1 mm diameter glass beads, under laminar or turbulent flow (porous media Reynolds number = 20 and 200 respectively). The morphology and dynamic behavior reflected those of biofilms grown in the open flow cells. The laminar biofilm was relatively uniform and after 23 d had inundated the majority of the pore spaces. Under turbulent flow the biofilm accumulated primarily in protected regions at contact points between the beads and formed streamers that trailed from the leeward face. Both biofilms caused a 2 to 3-fold increase in friction factor and in both cases there were sudden reductions in friction factor followed by rapid recovery, suggesting periodic sloughing and regrowth events. Time-lapse microscopy revealed that under both laminar and turbulent conditions biofilms flowed over the surface of the porous media. In some instances ripple structures formed. The velocity of biofilm flow was on the order of 10 μm h^?1 in the turbulent flow cell and 1.0 μm h^?1 in the laminar flow cell.     

GDP Induces PANC-1 Human Pancreatic Cancer Cell Death Preferentially under Nutrient Starvation by Inhibiting PI3K/Akt/mTOR/Autophagy Signaling Pathway

Pancreatic tumors are hypovascular, which leads to a poor nutrient supply to support the aggressively proliferating tumor cells. However, human pancreatic cancer cells have extreme resistance to nutrition starvation, which enables them to survive under severe metabolic stress conditions within the tumor microenvironment, a phenomenon known as “austerity” in cancer biology. Discovering agents which can preferentially inhibit the cancer cells’ ability to tolerate starvation conditions represents a new generation of anticancer agents. In this study, geranyl 2,4-dihydroxy-6-phenethylbenzoate (GDP), isolated from Boesenbergia pandurata rhizomes, exhibited potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition starvation conditions. GDP also possessed PANC-1 cell migration and colony formation inhibitory activities under normal nutrient-rich conditions. Mechanistically, GDP inhibited PI3K/Akt/mTOR/autophagy survival signaling pathway, leading to selective PANC-1 cancer cell death under the nutrition starvation condition. Therefore, GDP is a promising anti-austerity agent for drug development against pancreatic cancer.