The influence of phototrophic benthic biofilms on Cd, Cu, Ni, and Pb transport in permeable sediments

The effect of phototrophic biofilm activity on advective transport of cadmium (Cd), copper (Cu), nickel (Ni), and lead (Pb) in sandy sediments was examined using percolated columns. Cd and Ni in the effluent exhibited clear diel cycles in biofilm-containing columns, with concentrations at the end of dark periods exceeding those during illumination by up to 4.5- and 10-fold for Ni and Cd, respectively. Similar cycles were not observed for Pb or Cu. Breakthrough of the latter metals was greatly retarded and incomplete relative to Cd and Ni, and trends in biofilm treatments did not differ greatly from those in control columns. Inhibition of photosystem II by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) proved that diel cycles of Cd and Ni were controlled by oxygenic photosynthesis, and microsensor measurements showed that metal cycles closely matched metabolic activity-driven pH variations. The sorption edge pH for the sand/biofilm substrate followed the order Ni > Cd > Cu > Pb, and for Ni and Cd, was within the pH 7?C10 range observed in the biofilm-containing column. Adsorption dynamics over the light periods matched pH increases, but desorption during dark periods was incomplete and slower than the rate of change of pH. Over a diel cycle, desorption was less than adsorption, resulting in net binding of dissolved metals due to the biofilm metabolic activity. Extraction with selective reagents indicated that the adsorbed metals were readily exchangeable, and potentially bioavailable. Thus, phototrophic benthic biofilms can control the transport of some metals across the sand?Cwater interface, and processes in this very thin surficial layer should be considered when evaluating chemical fluxes in permeable sediments.     

Sorption and distribution of copper in unsaturated Pseudomonas putida CZ1 biofilms as determined by X-ray fluorescence microscopy

The spatial and temporal distribution of metals in unsaturated Pseudomonas putida CZ1 biofilms was determined using synchrotron-based X-ray fluorescence microscopy (XRF). It was found that Fe, Mn, and Ca were mainly distributed near the air-biofilm interface of a biofilm grown on 40 mM citrate, while there were two Fe-, Mn-, and Ca-rich layers within a biofilm grown on 10 mM citrate. The sorption of copper by biofilm grown in medium containing 10 mM citrate was rapid, with copper being found throughout the biofilm after only 1 h of exposure. Copper initially colocalized with Fe and Mn element layers in the biofilm and then precipitated in a 40-μm-thick layer near the air-biofilm interface when exposed for 12 h. Cu K-edge X-ray absorption near edge structure (XANES) analysis revealed that Cu was primarily bound with citrate within the biofilm, and the precipitate formed in the biofilm exposed to copper for 12 h was most similar to copper phosphate. LIVE/DEAD staining revealed that cells at the biofilm-membrane interface were mostly alive even when the copper concentration reached 80.5 mg copper g(-1) biomass. This suggests that the biofilm matrix provided significant protection for cells in this area. These results significantly improve our understanding of metal acquisition, transportation, and immobilization in unsaturated biofilm systems.     

Magnetic Resonance Imaging of Mass Transport and Structure Inside a Phototrophic Biofilm

The aim of this study was to utilize magnetic resonance imaging (MRI) to image structural heterogeneity and mass transport inside a biofilm which was too thick for photon based imaging. MRI was used to map water diffusion and image the transport of the paramagnetically tagged macromolecule, Gd-DTPA, inside a 2.5 mm thick cyanobacterial biofilm. The structural heterogeneity of the biofilm was imaged at resolutions down to 22 × 22 μm, enabling the impact of biofilm architecture on the mass transport of both water and Gd-DTPA to be investigated. Higher density areas of the biofilm correlated with areas exhibiting lower relative water diffusion coefficients and slower transport of Gd-DTPA, highlighting the impact of biofilm structure on mass transport phenomena. This approach has potential for shedding light on heterogeneous mass transport of a range of molecular mass molecules in biofilms.     

Integration of Raman microscopy, differential interference contrast microscopy, and attenuated total reflection Fourier transform infrared spectroscopy to investigate chlorhexidine spatial and temporal distribution in Candida albicans biofilms.

Two spectroscopic techniques, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and Raman microscopy (RM), were used to characterize transport of chlorhexidine digluconate (CHG) in Candida albicans (CA) biofilms. Different (volumetric) regions of the biofilm are sampled by these two vibrational spectroscopies making them complementary techniques. Simple mathematical models were developed to analyze ATR-FTIR and RM data to obtain an effective diffusion coefficient describing transport through CA biofilms. CA biofilms were composed primarily of yeast and hyphal forms, with some pseudohyphae. Upper regions of biofilms that had become confluent, (i.e., biofilms that completely covered the germanium (Ge) substratum) were composed primarily of a tangled mass of hyphae with openings between germtubes about 10 to 50 microm across. Quantitative analysis of ATR-FTIR kinetic data curves indicated that the effective diffusion coefficient for transport of CHG through confluent biofilms about 200-microm thick was reduced 0.1 to 0.3 times compared to the diffusion coefficient for CHG in water. Effective diffusion coefficients obtained from analysis of RM data were consistently higher than those indicated by ATR-FTIR data suggesting that transport is more hindered in regions near the base of the biofilm than in the outer layers. Analysis of both ATR-FTIR and RM data obtained from thicker films indicated that adsorption of CHG to biofilm components was responsible for a substantial portion of the transport limitation imposed by the biofilm. Comparison of ATR-FTIR and RM data for both types of biofilms indicated that sites of CHG adsorption were more concentrated in the interfacial region than in the bulk biofilm. Comparison of results for ATR-FTIR and RM measurements suggests that these relatively thick CA biofilms can be modeled, for purposes of predicting transport, approximately as a homogeneous thin planar sheet. Thus, these biofilms offer a relatively tractable model system for initial investigations of the relation between antimicrobial transport and kinetics of antimicrobial action.     

Electroactivity of phototrophic river biofilms and constitutive cultivable bacteria

Electroactivity is a property of microorganisms assembled in biofilms that has been highlighted in a variety of environments. This characteristic was assessed for phototrophic river biofilms at the community scale and at the bacterial population scale. At the community scale, electroactivity was evaluated on stainless steel and copper alloy coupons used both as biofilm colonization supports and as working electrodes. At the population scale, the ability of environmental bacterial strains to catalyze oxygen reduction was assessed by cyclic voltammetry. Our data demonstrate that phototrophic river biofilm development on the electrodes, measured by dry mass and chlorophyll a content, resulted in significant increases of the recorded potentials, with potentials of up to +120 mV/saturated calomel electrode (SCE) on stainless steel electrodes and +60 mV/SCE on copper electrodes. Thirty-two bacterial strains isolated from natural phototrophic river biofilms were tested by cyclic voltammetry. Twenty-five were able to catalyze oxygen reduction, with shifts of potential ranging from 0.06 to 0.23 V, cathodic peak potentials ranging from -0.36 to -0.76 V/SCE, and peak amplitudes ranging from -9.5 to -19.4 μA. These isolates were diversified phylogenetically (Actinobacteria, Firmicutes, Bacteroidetes, and Alpha-, Beta-, and Gammaproteobacteria) and exhibited various phenotypic properties (Gram stain, oxidase, and catalase characteristics). These data suggest that phototrophic river biofilm communities and/or most of their constitutive bacterial populations present the ability to promote electronic exchange with a metallic electrode, supporting the following possibilities: (i) development of electrochemistry-based sensors allowing in situ phototrophic river biofilm detection and (ii) production of microbial fuel cell inocula under oligotrophic conditions.     

Regulation of copper uptake and transport in intestinal cell monolayers by acute and chronic copper exposure

Adaptation to high and low copper intake in mammals depends on the cellular control of influx, efflux and storage mechanisms of cellular copper concentrations. In the present study, we used an intestinal cell line (Caco-2), grown in bicameral chambers to study the effect of equilibrium loading with copper. We analyzed (64)Cu uptake from the apical surface, intracellular metal (Cu, Zn, Fe) content, (64)Cu transport into the basal chamber, and total copper, zinc and iron in the basal chamber. We found that the (64)Cu uptake is saturable, shows a linear response phase up to 1.5 microM reaching a plateau at 4-6 microM extracellular Cu. Intracellular copper increased 21.6-fold, from 1.5 to 32.4 mM (at 0.2-20.2 microM extracellular copper respectively). The time course for (64)Cu uptake and transport was linear when the cells were incubated with different copper concentrations. Uptake increased 10-fold when intracellular copper concentration was raised. Fluxes were lowest at 1.5 mM and highest at 32.4 mM Cu intracellular copper (2.03 and 20. 98 pmole (64)Cu insert(-1) h(-1), respectively). The apical-to-basolateral copper transfer rate was lower at 32.4 mM as compared to 1.5 mM intracellular copper (0.55-1.95 pmole (64)Cu insert(-1) h(-1), respectively). The total copper in the basal chamber increased 4.2-fold (from 3.04 to 12.85 pmole Cu insert(-1) h(-1)) when the intracellular copper concentration was raised. If cells are preincubated in a low copper medium most of the newly incorporated copper (64%) is transferred to the basolateral compartment. In contrast, under preloading with high copper concentration, only 4% of the fresh copper is transferred to the basal chamber; however, the intracellular copper contribution to this chamber increases by 4.2-fold. Thus, the process results in an increase in both storage and intracellular-to-basolateral flux of copper. In summary, our results indicate that copper fluxes from apical-to-cell and apical-to-basolateral domains are affected by intracellular copper concentration suggesting that mechanisms of copper transport involved in cellular adaptation to low and high copper exposure are different.     

Biofilm shows spatially stratified metabolic responses to contaminant exposure

Biofilms are core to a range of biological processes, including the bioremediation of environmental contaminants. Within a biofilm population, cells with diverse genotypes and phenotypes coexist, suggesting that distinct metabolic pathways may be expressed based on the local environmental conditions in a biofilm. However, metabolic responses to local environmental conditions in a metabolically active biofilm interacting with environmental contaminants have never been quantitatively elucidated. In this study, we monitored the spatiotemporal metabolic responses of metabolically active Shewanella oneidensis MR‐1 biofilms to U(VI) (uranyl, UO₂ ²⁺) and Cr(VI) (chromate, CrO₄ ^2?) using non‐invasive nuclear magnetic resonance imaging (MRI) and spectroscopy (MRS) approaches to obtain insights into adaptation in biofilms during biofilm‐contaminant interactions. While overall biomass distribution was not significantly altered upon exposure to U(VI) or Cr(VI), MRI and spatial mapping of the diffusion revealed localized changes in the water diffusion coefficients in the biofilms, suggesting significant contaminant‐induced changes in structural or hydrodynamic properties during bioremediation. Finally, we quantitatively demonstrated that the metabolic responses of biofilms to contaminant exposure are spatially stratified, implying that adaptation in biofilms is custom‐developed based on local microenvironments.     

Determination of pollutant diffusion coefficients in naturally formed biofilms using a single tube extractive membrane bioreactor

A novel technique has been used to determine the effective diffusion coefficients for 1,1,2-trichloroethane (TCE), a nonreacting tracer, in biofilms growing on the external surface of a silicone rubber membrane tube during degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 and monochlorobenzene (MCB) by Pseudomonas JS150. Experiments were carried out in a single tube extractive membrane bioreactor (STEMB), whose configuration makes it possible to measure the transmembrane flux of substrates. A video imaging technique (VIT) was employed for in situ biofilm thickness measurement and recording. Diffusion coefficients of TCE in the biofilms and TCE mass transfer coefficients in the liquid films adjacent to the biofilms were determined simultaneously using a resistances-in-series diffusion model. It was found that the flux and overall mass transfer coefficient of TCE decrease with increasing biofilm thickness, showing the importance of biofilm diffusion on the mass transfer process. Similar fluxes were observed for the nonreacting tracer (TCE) and the reactive substrates (MCB or DCE), suggesting that membrane-attached biofilm systems can be rate controlled primarily by substrate diffusion. The TCE diffusion coefficient in the JS150 biofilm appeared to be dependent on biofilm thickness, decreasing markedly for biofilm thicknesses of >1 mm. The values of the TCE diffusion coefficients in the JS150 biofilms <1-mm thick are approximately twice those in water and fall to around 30% of the water value for biofilms >1-mm thick. The TCE diffusion coefficients in the GJ10 biofilms were apparently constant at about the water value. The change in the diffusion coefficient for the JS150 biofilms is attributed to the influence of eddy diffusion and convective flow on transport in the thinner (<1-mm thick) biofilms.     

Diffusion in Pseudomonas aeruginosa biofilms: a pulsed field gradient NMR study

A Pseudomonas aeruginosa biofilm is studied with pulsed field gradient echo nuclear magnetic resonance. Although not all spectral components are assigned yet, the experimental results show that a biofilm consists of components with very different diffusion coefficients. The various biofilm components that give motionally narrowed 1H NMR signals, can be grouped into five classes with diffusion coefficients, ranging from 2 x 10(-9) to 1 x 10(-13) m2 s-1. Investigation of the diffusion behavior of water in the biofilm shows three fractions with different diffusion coefficients. Besides the highly mobile bulk water at least two other fractions with much lower diffusion coefficients are detected. It is shown that one of the fractions with the low diffusion coefficient probably arises from intracellular water. Also for another component of the biofilm, glycerol, three fractions with diffusion coefficients that differ more than a factor ten are detected. Also a group of signals exists which result from practically immobile components.     

Biosorption of copper(II) ions onto powdered waste sludge in a completely mixed fed-batch reactor: estimation of design parameters

Biosorption of Cu(II) ions onto pre-treated powdered waste sludge (PWS) was investigated using a fed-batch operated completely mixed reactor. Fed-batch adsorption experiments were performed by varying the feed flow rate ( 0.075-0.325 l h(-1)), feed copper (II) ion concentrations (50-300 mg l(-1)) and the amount of adsorbent (1-6 g PWS) using fed-batch operation. Breakthrough curves describing the variations of effluent copper ion concentrations with time were determined for different operating conditions. Percent copper ion removals from the aqueous phase decreased, but the biosorbed (solid phase) copper ion concentrations increased with increasing the feed flow rate and Cu(II) concentration. A modified Bohart-Adams equation was used to determine the biosorption capacity of PWS and the rate constant for Cu(II) ion biosorption. Adsorption rate constant in fed-batch operation was an order of magnitude larger than those obtained in adsorption columns because of elimination of mass transfer limitations encountered in the column operations while the biosorption capacity of PWS was comparable with powdered activated (PAC) in column operations. Therefore, a completely mixed reactor operated in fed-batch mode was proven to be more advantageous as compared to adsorption columns due to better contact between the phases yielding faster adsorption rates.     

Regulation of copper absorption by copper availability in the Caco-2 cell intestinal model

Relatively little is known about the individual steps in intestinal copper absorption and whether or how they may be regulated. Polarized Caco-2 cell monolayers with tight junctions offer an already tested model in which to study intestinal metal transport. This model was used to examine potential effects of cellular copper availability on copper absorption. Uptake and transport were determined on application of (64)Cu(II) to the brush border. In the range of 0.2-2 micro M, uptake was dose dependent and was approximately 20% of dose/90 min. Overall transport of (64)Cu across the basolateral surface was approximately 0.3%. When cellular copper levels were depleted 40% by 18-h pretreatment with the specific copper chelator triethylenetetraamine, uptake and overall transport were markedly increased, going to 80 and 65% of dose, respectively. Cellular retention of (64)Cu fell fourfold, from 6 to 1.5%. Depletion of copper with the chelator was rapid and preceded initial changes in uptake and overall transport by 4 h. A lesser depletion of cellular copper (13%) failed to enhance copper uptake but doubled the rate of overall transport, as measured with (64)Cu and by atomic absorption. As previously reported, preexposure of the cells to excess copper (10 micro M, 18 h) also enhanced copper uptake ( approximately 3-fold). In contrast, ascorbate (10-1,000 micro M) failed to significantly alter uptake and transport of 1 micro M (64)Cu. Our findings are consistent with the concepts that, in the low physiological range, copper availability alters the absorption capacity of the intestine to support whole body homeostasis and that basolateral transport is more sensitively regulated than uptake.     

Analysis of Structural and Physiological Profiles To Assess the Effects of Cu on Biofilm Microbial Communities

We investigated the effects of copper on the structure and physiology of freshwater biofilm microbial communities. For this purpose, biofilms that were grown during 4 weeks in a shallow, slightly polluted ditch were exposed, in aquaria in our laboratory, to a range of copper concentrations (0, 1, 3, and 10 μM). Denaturing gradient gel electrophoresis (DGGE) revealed changes in the bacterial community in all aquaria. The extent of change was related to the concentration of copper applied, indicating that copper directly or indirectly caused the effects. Concomitantly with these changes in structure, changes in the metabolic potential of the heterotrophic bacterial community were apparent from changes in substrate use profiles as assessed on Biolog plates. The structure of the phototrophic community also changed during the experiment, as observed by microscopic analysis in combination with DGGE analysis of eukaryotic microorganisms and cyanobacteria. However, the extent of community change, as observed by DGGE, was not significantly greater in the copper treatments than in the control. Yet microscopic analysis showed a development toward a greater proportion of cyanobacteria in the treatments with the highest copper concentrations. Furthermore, copper did affect the physiology of the phototrophic community, as evidenced by the fact that a decrease in photosynthetic capacity was detected in the treatment with the highest copper concentration. Therefore, we conclude that copper affected the physiology of the biofilm and had an effect on the structure of the communities composing this biofilm.     

Copper(II)-cis,cis-1,3,5-triaminocyclohexane complex-promoted hydrolysis of dipeptides: kinetic, speciation and structural studies

The hydrolysis of glycylglycine (GylGly), glycyl-L-leucine (GlyLeu), L-leucylglycine (LeuGly) and glycyl-DL-serine (GlySer) promoted by a copper(II)- cis, cis-1,3,5-triaminocyclohexane complex [Cu(II)TACH] was investigated at 70 degrees C and pH 7-10, using HPLC. The observed pseudo-first-order rate constants (k(obs)) and rate enhancing factors (REF) were as follows: 4.1x10(-3 )h(-1)(REF=23) for GylGly, 1.6x10(-3 )h(-1)(REF=21) for GlyLeu, 5.1x10(-3 )h(-1)(REF=64) for LeuGly and 9.2x10(-2 )h(-1)(REF=47) for GlySer [pH 8.1, dipeptide 2 mM, copper(II) 2 mM and TACH 2 mM]. Based on the pH dependence and dipeptide concentration dependence of the initial rates and speciation of the Cu(II)-TACH-dipeptide system at 25 degrees C and I=0.1, the reactions proceed via the formation of a ternary complex [Cu(TACH)(dipeptide)](+) as an intermediate followed by OH(-)-dependent and OH(-)-independent paths to give amino acid(s). GylGly, GlyLeu and LeuGly preferred the OH(-)-dependent path, while GlySer preferred the OH(-)-independent path. The latter can be explained by the intramolecular attack of the amide carbonyl group coordinated with its oxygen atom by the OH group in the serine residue. The X-ray crystal structure of [Cu(TACH)(GlyGly)]BPh(4).MeOH confirmed that GlyGly coordinates to copper(II) ion with its terminal amino N and amide O atoms. The crystal structures of [Cu(TACH)(Gly)]BPh(4) and [Cu(2)(TACH)(2)(OH)(2)](ClO(4))(2).NaClO(4).H(2)O are also reported.     

Persister cells, the biofilm matrix and tolerance to metal cations in biofilm and planktonic Pseudomonas aeruginosa

In this study, we examined Pseudomonas aeruginosa ATCC 27853 biofilm and planktonic cell susceptibility to metal cations. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) required to eradicate 100% of the planktonic population (MBC 100), and the minimum biofilm eradication concentration (MBEC) were determined using the MBEC trade mark-high throughput assay. Six metals - Co(2+), Ni(2+), Cu(2+), Zn(2+), Al(3+) and Pb(2+)- were each tested at 2, 4, 6, 8, 10 and 27 h of exposure to biofilm and planktonic cultures grown in rich or minimal media. With 2 or 4 h of exposure, biofilms were approximately 2-25 times more tolerant to killing by metal cations than the corresponding planktonic cultures. However, by 27 h of exposure, biofilm and planktonic bacteria were eradicated at approximately the same concentration in every instance. Viable cell counts evaluated at 2 and 27 h of exposure revealed that at high concentrations, most of the metals assayed had killed greater than 99.9% of biofilm and planktonic cell populations. The surviving cells were propogated in vitro and gave rise to biofilm and planktonic cultures with normal sensitivity to metals. Further, retention of copper by the biofilm matrix was investigated using the chelator sodium diethlydithiocarbamate. Formation of visible brown metal-chelates in biofilms treated with Cu(2+) suggests that the biofilm matrix may coordinate and sequester metal cations from the aqueous surroundings. Overall, our data suggest that both metal sequestration in the biofilm matrix and the presence of a small population of 'persister' cells may be contributing factors in the time-dependent tolerance of both planktonic cells and biofilms to high concentrations of metal cations.     

Adsorptive removal of copper and nickel ions from water using chitosan coated PVC beads

A new biosorbent was developed by coating chitosan, a naturally and abundantly available biopolymer, on to polyvinyl chloride (PVC) beads. The biosorbent was characterized by FTIR spectra, porosity and surface area analyses. Equilibrium and column flow adsorption characteristics of copper(II) and nickel(II) ions on the biosorbent were studied. The effect of pH, agitation time, concentration of adsorbate and amount of adsorbent on the extent of adsorption was investigated. The experimental data were fitted to Langmuir and Freundlich adsorption isotherms. The data were analyzed on the basis of Lagergren pseudo first order, pseudo-second order and Weber-Morris intraparticle diffusion models. The maximum monolayer adsorption capacity of chitosan coated PVC sorbent as obtained from Langmuir adsorption isotherm was found to be 87.9 mg g(-1) for Cu(II) and 120.5 mg g(-1) for Ni(II) ions, respectively. In addition, breakthrough curves were obtained from column flow experiments. The experimental results demonstrated that chitosan coated PVC beads could be used for the removal of Cu(II) and Ni(II) ions from aqueous medium through adsorption.     

Nitrosocyanin, a red cupredoxin-like protein from Nitrosomonas europaea

Nitrosocyanin (NC), a soluble, red Cu protein isolated from the ammonia-oxidizing autotrophic bacterium Nitrosomonas europaea, is shown to be a homo-oligomer of 12 kDa Cu-containing monomers. Oligonucleotides based on the amino acid sequence of the N-terminus and of the C-terminal tryptic peptide were used to sequence the gene by PCR. The translated protein sequence was significantly homologous with the mononuclear cupredoxins such as plastocyanin, azurin, or rusticyanin, the type 1 copper-binding region of nitrite reductase, and the binuclear CuA binding region of N(2)O reductase or cytochrome oxidase. The gene for NC contains a leader sequence indicating a periplasmic location. Optical bands for the red Cu center at 280, 390, 500, and 720 nm have extinction coefficients of 13.9, 7.0, 2.2, and 0.9 mM(-1), respectively. The reduction potential of NC (85 mV vs SHE) is much lower than those for known cupredoxins. Sequence alignments with homologous blue copper proteins suggested copper ligation by Cys95, His98, His103, and Glu60. Ligation by these residues (and a water), a trimeric protein structure, and a cupredoxin beta-barrel fold have been established by X-ray crystallography of the protein [Lieberman, R. L., Arciero, D. M., Hooper, A. B., and Rosenzweig, A. C. (2001) Biochemistry 40, 5674-5681]. EPR spectra of the red copper center indicated a Cu(II) species with a g(parallel) of 2.25 and an A(parallel) of 13.8 mT (144 x 10(-4) cm(-1)), typical of Cu in a type 2 copper environment. NC is the first example of a type 2 copper center in a cupredoxin fold. The open coordination site and type 2 copper suggest a possible catalytic rather than electron transfer function.     

A study of acetic acid production by immobilized Acetobacter Cells: Oxygen transfer

The immobilization of living Acetobacter cells by adsorption onto a large-surface-area ceramic support was studied in a pulsed flow reactor. The high oxygen transfer capability of the reactor enabled acetic acid production rates up to 10.4 g L(-1) h(-1) to be achieved. Using a simple mathematical model incorporating both internal and external mass transfer coefficients, it was shown that oxygen transfer in the microbial film controls the reactor productivity.     

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.     

Local macromolecule diffusion coefficients in structurally non-uniform bacterial biofilms using fluorescence recovery after photobleaching (FRAP)

Pure culture Pseudomonas putida biofilms were cultivated under controlled conditions to a desired overall biofilm thickness, then employed within classical half-cell diffusion chambers to estimate, from transient solute concentrations, the effective diffusion coefficient for several macromolecules of increasing molecular weight and molecular complexity. Results of traditional half-cell studies were found to be erroneous due to the existence of microscopic water channels or crevasses that perforate the polysaccharidic gel matrix of the biofilm, sometimes completely to the supporting substratum. Thus, half-cell devices measure a composite transfer coefficient that may overestimate the true, local flux of solutes in the biofilm polysaccharide gel matrix. An alternative analytical technique was refined to determine the local diffusion coefficients on a micro-scale to avoid the errors created by the biofilm architectural irregularities. This technique is based upon the Fluorescence Return After Photobleaching (FRAP), which allows image analysis observation of the transport of fluorescently labeled macromolecules as they migrate into a micro-scale photobleached zone. The technique can be computerized and allows one to map the local diffusion coefficients of various solute molecules at different horizontal planes and depths in a biofilm. These mappings also indirectly indicate the distribution of water channels in the biofilm, which was corroborated independently by direct microscopic observation of the settling of fluorescently-labeled latex spheres within the biofilm. Fluorescence return after photobleaching results indicate a significant reduction in the solute transport coefficients in biofilm polymer gel vs. the same value in water, with the reduction being dependent on solute molecule size and shape.