Modeling biofilm accumulation and mass transport in a porous medium under high substrate loading

A packed bed biofilm reactor inoculated with pure culture Pseudomonas aeruginosa was run under high substrate loading and constant flow rate conditions. The 3.1-cm-diameter cylindrical reactor was 5 cm in length and packed with 1-mm glass beads. Daily observations of biofilm thickness, influent and effluent glucose substrate concentration, and effluent dissolved and total organic carbon were made during the 13-day experiment. Biofilm thickness appeared to rech quasi-steady-state condition after 10 days. A published biofilm process simulation program (AQUASIM) was used to analyze experimental data. Comparison of observed and simulated variables revealed three distinct phases of biofilm accumulation during the experiment: an initial phase, a growth phase, and a mature biofilm phase. Different combinations of biofilm and mass transport process variables were found to be important during each phase. Biofilm detachment was highly correlated with shear at the biofilm surface during all three phases of biofilm development. (c) 1995 John Wiley & Sons, Inc.     

A novel approach to investigate tissue-specific trinucleotide repeat instability

Background

In Huntington's disease (HD), an expanded CAG repeat produces characteristic striatal neurodegeneration. Interestingly, the HD CAG repeat, whose length determines age at onset, undergoes tissue-specific somatic instability, predominant in the striatum, suggesting that tissue-specific CAG length changes could modify the disease process. Therefore, understanding the mechanisms underlying the tissue specificity of somatic instability may provide novel routes to therapies. However progress in this area has been hampered by the lack of sensitive high-throughput instability quantification methods and global approaches to identify the underlying factors.

Results

Here we describe a novel approach to gain insight into the factors responsible for the tissue specificity of somatic instability. Using accurate genetic knock-in mouse models of HD, we developed a reliable, high-throughput method to quantify tissue HD CAG repeat instability and integrated this with genome-wide bioinformatic approaches. Using tissue instability quantified in 16 tissues as a phenotype and tissue microarray gene expression as a predictor, we built a mathematical model and identified a gene expression signature that accurately predicted tissue instability. Using the predictive ability of this signature we found that somatic instability was not a consequence of pathogenesis. In support of this, genetic crosses with models of accelerated neuropathology failed to induce somatic instability. In addition, we searched for genes and pathways that correlated with tissue instability. We found that expression levels of DNA repair genes did not explain the tissue specificity of somatic instability. Instead, our data implicate other pathways, particularly cell cycle, metabolism and neurotransmitter pathways, acting in combination to generate tissue-specific patterns of instability.

Conclusion

Our study clearly demonstrates that multiple tissue factors reflect the level of somatic instability in different tissues. In addition, our quantitative, genome-wide approach is readily applicable to high-throughput assays and opens the door to widespread applications with the potential to accelerate the discovery of drugs that alter tissue instability.     

A microscale model of bacterial and biofilm dynamics in porous media

A microscale model for the transport and coupled reaction of microbes and chemicals in an idealized two-dimensional porous media has been developed. This model includes the flow, transport, and bioreaction of nutrients, electron acceptors, and microbial cells in a saturated granular porous media. The fluid and chemicals are represented as a continuum, but the bacterial cells and solid granular particles are represented discretely. Bacterial cells can attach to the particle surfaces or be advected in the bulk fluid. The bacterial cells can also be motile and move preferentially via a run and tumble mechanism toward a chemoattractant. The bacteria consume oxygen and nutrients and alter the profiles of these chemicals. Attachment of bacterial cells to the soil matrix and growth of bacteria can change the local permeability. The coupling of mass transport and bioreaction can produce spatial gradients of nutrients and electron acceptor concentrations. We describe a numerical method for the microscale model, show results of a convergence study, and present example simulations of the model 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.     

Quantification of biofilm accumulation by an optical approach

Methods for non-invasive, in situ, measurements of biofilm optical density and biofilm optical thickness were evaluated based on Pseudomonas aeruginosa experiments. Biofilm optical density, measured as intensity reduction of a light beam transmitted through the biofilm, correlates with biofilm mass, measured as total carbon and as cell mass. The method is more sensitive and less labor intensive than other commonly used methods for determining extent of biofilm mass accumulation. Biofilm optical thickness, measured by light microscopy, is translated into physical thickness based on biofilm refraction measurements. Biofilm refractive index was found to be close to the refractive index of water. The P. aeruginosa biofilms studied reached a pseudo steady state in less than a week, with stable liquid phase substrate, cell and TOC concentrations and average biofilm thickness. True steady state was, however, not reached as both biofilm density and roughness were still increasing after 3 weeks.     

Significance of electrophoretic mobility distribution to bacterial transport in granular porous media

A method is developed to obtain the electrophoretic mobility distribution of colloidal particles by microelectrophoresis. The results demonstrate that for small particles (< 1 microm), the experimental mobility distribution must be deconvoluted to remove the effect of the random Brownian motion so that the electrophoretic mobility distribution can be obtained. For bacteria-sized particles (on the order of 1 microm or larger), the random Brownian motion is not significant, and the experimental mobility distribution represents the electrophoretic mobility distribution. The significance of the electrophoretic mobility distribution to bacterial transport is demonstrated through comparison between experimental and theoretical values of collision efficiency. Using the extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the electrophoretic mobility distribution of bacteria is transformed to the distribution of collision efficiencies. For strain Comamonas sp. DA001, the predicted collision efficiency values span orders of magnitude, indicating that variation of surface charge density in a monoclonal bacterial population is a cause for the orders of magnitude variation of experimentally determined collision efficiencies. However, despite the fact that the predicted and experimental alpha distributions overlap, the match is not adequate. This inadequacy is ascribed to inability to probe heterogeneity of bacterial surface hydrophobicity, and the inability of the DLVO theory to quantitatively model particle deposition.     

A biofilm model developed to investigate survival and disinfection of Mycobacterium mucogenicum in potable water

Water in healthcare environments can be a source for healthcare-associated infections (HAI). However, information on the exposure risk to opportunistic pathogens in potable water distribution systems (PWDS) is lacking. Laboratory studies characterizing the interaction of opportunistic pathogens with biofilms are needed to understand their role in water systems within healthcare facilities. A stable, repeatable, PWDS multi-species biofilm model comprising Sphingomonas paucimobilis, Methylobacterium sp., Delftia acidovorans, and Mycobacterium mucogenicum was developed in the CDC Biofilm Reactor (CBR), reaching 6 log₁₀ CFU cm^?2 within 6 days. The model was used to investigate the interaction of the opportunistic pathogen M. mucogenicum with the other species, and to determine the efficacy of monochloramine (NH₂Cl) as a disinfectant against 2-week-old biofilms. Addition of 1 or 2 mg l^?1 NH₂Cl resulted in the same or an increased log density of viable M. mucogenicum in the biofilm while inactivating some of the Proteobacteria. Although M. mucogenicum preferentially resided in the biofilm, NH₂Cl exposure caused release of viable M. mucogenicum from the biofilm into the water. Additional studies with this model should determine if sodium hypochlorite has a comparative effect and if other nontuberculous mycobacteria (NTM) respond to NH₂Cl similarly.     

A novel in silico approach to identify potential therapeutic targets in human bacterial pathogens

In recent years, genome-sequencing projects of pathogens and humans have revolutionized microbial drug target identification. Of the several known genomic strategies, subtractive genomics has been successfully utilized for identifying microbial drug targets. The present work demonstrates a novel genomics approach in which codon adaptation index (CAI), a measure used to predict the translational efficiency of a gene based on synonymous codon usage, is coupled with subtractive genomics approach for mining potential drug targets. The strategy adopted is demonstrated using respiratory pathogens, namely, Streptococcus pneumoniae and Haemophilus influenzae as examples. Our approach identified 8 potent target genes (Streptococcus pneumoniae?C2, H. influenzae?C6), which are functionally significant and also play key role in host-pathogen interactions. This approach facilitates swift identification of potential drug targets, thereby enabling the search for new inhibitors. These results underscore the utility of CAI for enhanced in silico drug target identification.     

A mixture approach to investigate interstitial growth in engineering scaffolds

Controlling biological growth within a cell-laden polymeric scaffold is a critical challenge in the tissue engineering community. Indeed, construct growth must often be balanced with scaffold degradation and is often coupled to varying degrees of deformation that originate from swelling, external forces and the effects of confinement. These factors have been shown to affect growth in many ways, but to date, our understanding is mostly qualitative. While cell sensing, molecular transport and scaffold/tissue interactions are believed to be important players, it will be critical to quantify, predict and control these effects in order to eventually optimize tissue growth in the laboratory. The aim of this paper was thus to provide a theoretical framework to better understand how the scaffold-mediated mechanisms of transport, deposition (and possibly degradation) and elasticity affect the overall growth of a tissue subjected to finite deformations. We propose a formulation in which the macroscopic evolutions in tissue size, density as well as the appearance of residual stresses can be directly related to changes in internal composition by considering three fundamental principles: mechanical equilibrium, chemical equilibrium and molecular incompressibility. The resulting model allows us to pay particular attention to features that are critical to the interaction between growth and deformation: osmotic pressure and swelling, the strain mismatch between old and newly deposited material as well as the mechano-sensitive cell-mediated production. We show that all of these phenomena may indeed strongly affect the overall growth of a construct under finite deformations.     

A simple in vivo approach to investigate invasive trophoblast cells

Intrauterine trophoblast cell invasion is an essential part of hemochorial placentation. Aberrant trophoblast cell invasion has been associated with pathologies including preeclampsia and fetal growth restriction. In this study, we describe an in vivo method to assess trophoblast cell invasion using a transgenic rat model, constitutively expressing heat stable human placental alkaline phosphatase (Rosa 26 promoter driven human placental alkaline phosphatase, R26-hAP). Wild-type female Fischer 344 inbred rats were mated with hemizygous R26-hAP transgenic male Fischer 344 rats and sacrificed during the second half of pregnancy. Heat stable alkaline phosphatase (AP) activity associated with the invasive transgenic trophoblast cells was monitored in the wild-type uterine mesometrial compartment and used as an index of trophoblast cell invasion. The expression pattern of cytokeratins by invasive trophoblast cells mimicked the uterine mesometrial distribution of AP activity. Trophoblast cell invasion exhibited a gestation-dependent profile with peak invasion between days 18-20 of pregnancy. In summary, we have devised a simple in vivo method for assessing intrauterine trophoblast cell invasion. This technique should facilitate the discovery of endogenous regulatory mechanisms controlling trophoblast cell invasion and should represent an effective method of testing the impact of various environmental stressors on an essential part of hemochorial placentation.     

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.     

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.     

A communal bacterial adhesin anchors biofilm and bystander cells to surfaces

While the exopolysaccharide component of the biofilm matrix has been intensively studied, much less is known about matrix-associated proteins. To better understand the role of these proteins, we undertook a proteomic analysis of the V. cholerae biofilm matrix. Here we show that the two matrix-associated proteins, Bap1 and RbmA, perform distinct roles in the biofilm matrix. RbmA strengthens intercellular attachments. In contrast, Bap1 is concentrated on surfaces where it serves to anchor the biofilm and recruit cells not yet committed to the sessile lifestyle. This is the first example of a biofilm-derived, communally synthesized conditioning film that stabilizes the association of multilayer biofilms with a surface and facilitates recruitment of planktonic bystanders to the substratum. These studies define a novel paradigm for spatial and functional differentiation of proteins in the biofilm matrix and provide evidence for bacterial cooperation in maintenance and expansion of the multilayer biofilm.     

One-domain approach for studying multiphase transport phenomena in biofilm growing systems

The one-domain approach (ODA) was used as an alternative to solve fluid–biofilm interfacial behavior in a 2-D model for diffusion–reaction–convection coupled with prediction of irregular growth of biofilms via a cellular automaton strategy. The simulations exhibited errors of <7% compared with the porosity of a previously reported capillary experimental system. Additionally, biofilm surface geometrical aspects were satisfactorily compared with reports of experimental and similar rigorously simulated benchmark systems. The method developed was applied to simulate typical biofilm systems predicting recirculation flow patterns, interface concentration profiles, and clogging of the inlet section of the capillary tube, which are phenomena that affect the efficiency of diverse biotechnological applications, including membrane bioreactors and biofilters. The ODA method applied to the governing equations of momentum and mass transfer combined with a cellular automaton algorithm is a suitable and straightforward approach for modeling solid-state fermentation at different sophistication levels.     

A novel approach for whey recycling in production of bacterial insecticides

A simplified approach was devised to recycle sweet whey in production of spore-δ-endotoxin complex from certain entomopathogenic varieties ofBacillus thuringiensis Berliner. The process suggested aimed at the protection of the environment through dual channels namely biological oxygen demand (BOD) reduction of the byproduct under investigation and its incorporation in a microbial fermentation for production of pollution-free biological insecticides. The sweet whey could be used successfully for endotoxin production as complete fermentation media both as such and with simple treatments. Supplementation of whey media with ground leguminous seeds and fodder yeast resulted in marked increase in the yields of endotoxin produced but the toxicity was not increased proportionnally. Standard biological assays revealed high efficiency of certain strains ofB.t. var.entomocidus, kurstaki andgalleriae in producing endotoxins highly active against 3rd instar larvae ofSpodoptera littoralis Boisduval,Spodoptera exigua Hübner andHeliothis armigera Hübner. The suggested approach and the findings obtained are discussed in view of their application feasibilities.

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Résumé Une méthode simplifiée a été mise au point pour recycler le petit lait dans la production du complexe spore-δ-endotoxine de certaines variétés entomopathogènes deBacillus thuringiensis Berliner. Le procédé proposé a pour but la protection de l'environnement par un double canal chimique, c'est-à-dire la réduction de la demande biologique en oxygène du sous produit étudié et son incorporation dans une fermentation microbienne pour la fabrication d'insecticides biologiques non polluants. Le petit lait peut être employé avec succès pour la production d'endotoxine à la fois en tant que milieux complets de fermentation et avec des traitements simples. L'addition aux milieux à base de petit lait de graines de légumineuses et de levure alimentaire aboutit à une augmentation sensible des rendements en endotoxine mais la toxicité n'est pas accrue en proportion. Des essais biologiques normalisés montrent une activité élevée de certaines souches deB.t. var.entomocidus, kurstaki etgalleriae qui produisent des endotoxines très actives à l'égard des larves de 3e stage deSpodoptera littoralis Boisduval,Spodoptera exigua Hübner etHeliothis armigera Hübner. La méthode proposée et les résultats obtenus sont discutés en vue de leur faisabilité d'application.

     

Optically transparent porous medium for nondestructive studies of microbial biofilm architecture and transport dynamics

We describe a novel and noninvasive, microscopy-based method for visualizing the structure and dynamics of microbial biofilms, individual fluorescent microbial cells, and inorganic colloids within a model porous medium. Biofilms growing in flow cells packed with granules of an amorphous fluoropolymer could be visualized as a consequence of refractive index matching between the solid fluoropolymer grains and the aqueous immersion medium. In conjunction with the capabilities of confocal microscopy for nondestructive optical sectioning, the use of amorphous fluoropolymers as a solid matrix permits observation of organisms and dynamic processes to a depth of 2 to 3 mm, whereas sediment biofilms growing in sand-filled flow cells can only be visualized in the region adjacent to the flow cell wall. This method differs fundamentally from other refractive index-matching applications in that optical transparency was achieved by matching a solid phase to water (and not vice versa), thereby permitting real-time microscopic studies of particulate-containing, low-refractive-index media such as biological and chromatographic systems.     

An approach to identifying novel substrates of bacterial arylamine N-acetyltransferases

Arylamine N-acetyltransferases (NATs) catalyse the acetylation of arylamine, arylhydrazine and arylhydroxylamine substrates by acetyl Coenzyme A. NAT has been discovered in a wide range of eukaryotic and prokaryotic species. Although prokaryotic NATs have been implicated in xenobiotic metabolism, to date no endogenous role has been identified for the arylamine N-acetyl transfer reaction in prokaryotes. Investigating the substrate specificity of these enzymes is one approach to determining a possible endogenous role for prokaryotic NATs. We describe an accurate and efficient assay for NAT activity that is suitable for high-throughput screening of potential NAT ligands. This assay has been utilised to identify novel substrates for pure NAT from Salmonella typhimurium and Mycobacterium smegmatis which show a relationship between the lipophilicity of the arylamine and its activity as a substrate. The lipophilic structure/activity relationship observed is proposed to depend on the topology of the active site using docking studies of the crystal structures of these NAT isoenzymes. The evidence suggests an endogenous role of NAT in the protection of bacteria from aromatic and lipophilic toxins.