Strain stability in biological systems treating recalcitrant organic compounds

The availability of molecular probing technology in recent years has facilitated investigation of microbial community composition during bio-treatment of organic wastes. Particularly, it has allowed the study of microbial culture stability and correlation between stability and treatment performance. However, most studies to date have only addressed mixed cultures and there is limited information regarding single strain stability. Here we have investigated the microbial community dynamics in two bioreactors, each inoculated with a pure bacterial strain capable of degrading a recalcitrant substrate, namely Xanthobacter aut. GJ10 degrading 1,2-dichloroethane (DCE) and Burkholderia sp. JS150 degrading monochlorobenzene (MCB). Universal and strain specific 16S rRNA oligonucleotide probes were designed and used to follow strain stability. The bioreactor fed with DCE was functionally stable and the percentage of GJ10 cells in the community remained high (around 95% of total cells) throughout, even after introduction of foreign microorganisms. The bioreactor fed with MCB was also functionally stable, but in contrast to the DCE bioreactor, probing results revealed the disappearance of strain JS150 from the bioreactor within a week. The difference in behavior between the two systems is attributed to the specific pathway required to degrade DCE.     

Biofilm development in a membrane-aerated biofilm reactor: effect of flow velocity on performance

The effect of liquid flow velocity on biofilm development in a membrane-aerated biofilm reactor was investigated both by mathematical modeling and by experiment, using Vibrio natriegens as a test organism and acetate as carbon substrate. It was shown that velocity influenced mass transfer in the diffusion boundary layer, the biomass detachment rate from the biofilm, and the maximum biofilm thickness attained. Values of the overall mass transfer coefficient of a tracer through the diffusion boundary layer, the biofilm, and the membrane were shown to be identical during different experiments at the maximum biofilm thickness. Comparison of the results with published values of this parameter in membrane attached biofilms showed a similar trend. Therefore, it was postulated that this result might indicate the mechanism that determines the maximum biofilm thickness in membrane attached biofilms. In a series of experiments, where conditions were set so that the active layer of the membrane attached biofilm was located close to the membrane biofilm interface, it was shown that the most critical effect on process performance was the effect of velocity on biofilm structure. Biofilm thickness and effective diffusivity influenced reaction and diffusion in a complex manner such that the yield of biomass on acetate was highly variable. Consideration of endogenous respiration in the mathematical model was validated by direct experimental measurements of yield coefficients. Good agreement between experimental measurements of acetate and oxygen uptake rates and their prediction by the mathematical model was achieved.     

Microelectrode measurements of local mass transport rates in heterogeneous biofilms

Microelectrodes were used to measure oxygen profiles and local mass transfer coefficient profiles in biofilm clusters and interstitial voids. Both profiles were measured at the same location in the biofilm. From the oxygen profile, the effective diffusive boundary layer thickness (DBL) was determined. The local mass transfer coefficient profiles provided information about the nature of mass transport near and within the biofilm. All profiles were measured at three different average flow velocities, 0.62, 1.53, and 2.60 cm sec-1, to determine the influence of flow velocity on mass transport. Convective mass transport was active near the biofilm/liquid interface and in the upper layers of the biofilm, independent of biofilm thickness and flow velocity. The DBL varied strongly between locations for the same flow velocities. Oxygen and local mass transfer coefficient profiles collected through a 70 micrometer thick cluster revealed that a cluster of that thickness did not present any significant mass transport resistance. In a 350 micrometer thick biofilm cluster, however, the local mass transfer coefficient decreased gradually to very low values near the substratum. This was hypothetically attributed to the decreasing effective diffusivity in deeper layers of biofilms. Interstitial voids between clusters did not seem to influence the local mass transfer coefficients significantly for flow velocities of 1.53 and 2.60 cm sec-1. At a flow velocity of 0.62 cm sec-1, interstitial voids visibly decreased the local mass transfer coefficient near the bottom.     

Microbial dynamics in a continuous stirred tank bioreactor exposed to an alternating sequence of organic compounds

Microbial dynamics during aerobic biodegradation of an alternating mixture of organic compounds was investigated experimentally in a continuous stirred tank bioreactor (CSTB). A mathematical model describing this system was developed and tested using the experimental results. A model microbial culture consisting of Pseudomonas sp. JS150, a monochlorobenzene (MCB) degrader, and Xanthobacter autotrophicus GJ10, a 1,2-dichloroethane (DCE) degrader, each with exclusive degradation capabilities, was used. The CSTB was inoculated with both microbial strains and exposed to an alternating sequence of the two compounds at noninhibitory concentrations. Concentrations of each microbial strain, of each organic compound, and of degradation product evolved, as well as specific microbial activities via oxygen uptake tests, were monitored. Reduction of the residual DCE discharged from the bioreactor after an MCB to DCE transition was successfully achieved by continuously feeding a low flow of a concentrated solution of both compounds.     

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.     

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.     

Biofilm development in a membrane-aerated biofilm reactor: effect of intra-membrane oxygen pressure on performance

The effect on intra-membrane oxygen pressure at a constant carbon substrate loading rate on the development of biofilms of Vibrio natrigens in a membrane aerated biofilm reactor (MABR) was investigated experimentally and by mathematical modelling. A recently reported technique (Zhang et al., 1998. Biotechnol. Bioeng. 59: 80-89) for the in situ measurement of the substrate diffusion coefficients in a growing biofilm and the mass transfer coefficients in the boundary layer at the biofilm liquid interface was used. This aided the study of the effect of the heterogeneous biofilm structure and also improved the reliability of the model predictions. The different intra-membrane oxygen pressures used, 12.5, 25 and 50 kPa, with acetate as the carbon substrate, showed a marked effect on the initial biofilm growth rate, on acetate removal rate, particularly in thick biofilms and on biofilm structure. The model predicted the substrate limitation regimes, the location of the active biomass layer within the biofilms and the trends in oxygen uptake rate through the membrane into the biofilms. During the development of the biofilms, the biofilm thickness and the intra-membrane oxygen pressure were found to be the most important parameters influencing the MABR performance while the effect of biofilm structure was less marked.     

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.     

Membrane-attached biofilms for VOC wastewater treatment I: Novel in situ biofilm thickness measurement technique

This article reports a novel nondisruptive technique for measuring the thicknesses of membrane-attached biofilms in situ, using a single tube extractive membrane bioreactor (STEMB). The biodegradation of a toxic volatile organic compound (VOC) (1,2-dichloroethane [DCE]) by Xanthobacter autotrophicus GJ10 has been used as a model system to develop the technique. The results give information on the biomass-silicone rubber attachment phenomena, and on the development over time of biofilms growing on the silicone membrane, without disrupting operation. Experimental results are presented showing the evolution over time of biofilm thickness, and also the density of biofilms for four experimental runs. The hydrodynamic conditions on the biomedium side of the membrane were found to influence the initial attachment phenomena and subsequent biofilm growth. (c) 1995 John Wiley & Sons, Inc.     

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface‐averaging methods are used, position‐dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time‐dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two‐dimensional effective diffusion coefficient maps within Shewanella oneidensis MR‐1 biofilms using pulsed‐field gradient nuclear magnetic resonance methods, and used them to calculate surface‐averaged relative effective diffusion coefficient (D_rs) profiles. We found that (1) D_rs decreased from the top of the biofilm to the bottom, (2) D_rs profiles differed for biofilms of different ages, (3) D_rs profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D_rs profiles near the top of the biofilm, and (5) the D_rs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm. Biotechnol. Bioeng. 2010;106: 928–937. © 2010 Wiley Periodicals, Inc.     

Membrane-attached biofilms for VOC wastewater treatment. II: Effect of biofilm thickness on performance

This article reports a study of the performance of membrane-attached biofilms grown in a single tube extractive membrane bioreactor (STEMS) used for the treatment of a synthetic wastewater containing a toxic VOC (1,2-dichloroethane [DCE]). Mass balances show that complete mineralization of DCE was achieved, and that the biofilms were effective in reducing air stripping to negligible levels. Experimental results are presented showing the evolution over time of biofilm thickness and its influence on the flux of DCE across the membrane. It has been found that a trade-off exists between the positive influence of biofilms in reducing air-stripping of DCE, and the negative influence of biofilms in reducing DCE flux across the membrane. These considerations lead to an optimal biofilm thickness in the region of 200 to 400 mum being recommended for this system. (c) 1995 John Wiley & Sons, Inc.     

Quantitative analysis of biofilm thickness variability

The thickness variability of biofilms of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the binary population combination of these two species was quantified. The experimental method involved cryoembedding biofilms with a commercial tissue embedding agent, sectioning, and applying image analysis to construct thickness profiles along linear transects (up to 1 cm in length) across the substratum. Biofilms embedded and sectioned by this method were locally as thin as a single cell attached to the surface (<5 mum) and as thick as 1000 mum. Week-old biofilms of three different species compositions displayed distinct structural features as indicated by their mean thicknesses and by a roughness coefficient. Monopopulation biofilms of P. aeruginosa (29 mum mean thickness) or K. pneumoniae (100 mum mean thickness) were thinner than the binary population biofilm (400 mum mean thickness). A roughness coefficient developed in this investigation corroborated the qualitative visual characterization of P. aeruginosa biofilms as relatively uniformly thick (mean roughness coefficient 0.15), K. pneumoniae biofilms as patchy (mean roughness coefficient 1.14), and the binary population biofilm as intermediate (mean roughness coefficient 0.26). Whereas P. aeruginosa and binary population biofilms covered the substratum completely, significant areas of essentially bare substratum were apparent in K. pneumoniae biofilms. The patchiness of K. pneumoniae biofilms may be due to the fact that this organism is nonmotile. A spatial correlation analysis of the thickness data indicated that thickness measurements were still correlated even when separated by distances that exceeded the mean biofilm thickness. Cell aggregates, some of them hundreds of microns in size, were observed in the effluent of K. pneumoniae and binary population biofilm reactors. Measurements of thickness variability and other observations reported in this article provide a quantitative basis for analysis of microscale structural heterogeneity of biofilms. (c) 1995 John Wiley & Sons, Inc.     

Biofilm reactors: an experimental and modeling study of wastewater denitrification in fluidized-bed reactors of activated carbon particles

A fluidized-bed biofilm reactor using activated carbon particles of 1.69 mm diameter as the support for biomass growth and molasses as the carbon source is used for wastewater denitrification.The start-up of the reactor was successfully achieved in 1 week by using a liquor from garden soil leaching as the inoculum and a superficial velocity u(0) = 5u(mf). Typical biofilm thickness is 800 mum; therefore covered activated carbon particles have 3.3 mm in diameter.Reactor hydrodynamics was studied by tracer (KCl solution) experiments. The analysis based on residence time distribution theory involved a model with axial dispersion flow and tracer diffusion with linear adsorption inside the biofilm. Peclet numbers higher than 100 were found, allowing the plug flow assumption for the reactor model.Experimental profiles of nitrate and nitrite species were explained by a kinetic model of two consecutive zero-order reactions coupled with substrate diffusion inside the biofilm. Under the operating conditons used thick biofilms were obtained working in a diffusion-controlled regime.Comparison is made with results obtained in the same reactor with sand particles as the support for biomass growth. Activated carbon as the support has the following advantages: good adsorptive characteristics, homogeneous biofilm thickness along the reactor, and easy restart-up of the reactor. (c) 1992 John Wiley & Sons, Inc.     

A theoretical study on the effect of surface roughness on mass transport and transformation in biofilms

This modeling study evaluates the influence of biofilm geometrical characteristics on substrate mass transfer and conversion rates. A spatially two-dimensional model was used to compute laminar fluid flow, substrate mass transport, and conversion in irregularly shaped biofilms. The flow velocity above the biofilm surface was varied over 3 orders of magnitude. Numerical results show that increased biofilm roughness does not necessarily lead to an enhancement of either conversion rates or external mass transfer. The average mass transfer coefficient and Sherwood numbers were found to decrease almost linearly with biofilm area enlargement in the flow regime tested. The influence of flow, biofilm geometry and biofilm activity on external mass transfer could be quantified by Sh-Re correlations. The effect of biofilm surface roughness was incorporated in this correlation via area enlargement. Conversion rates could be best correlated to biofilm compactness. The more compact the biofilm, the higher the global conversion rate of substrate. Although an increase of bulk fluid velocity showed a large effect on mass transfer coefficients, the global substrate conversion rate per carrier area was less affected. If only diffusion occurs in pores and channels, then rough biofilms behave as if they were compact but having less biomass activity. In spite of the fact that the real biofilm area is increased due to roughness, the effective mass transfer area is actually decreased because only biofilm peaks receive substrate. This can be explained by the fact that in the absence of normal convection in the biofilm valleys, the substrate gradients are still largely perpendicular to the carrier. Even in the cases where convective transport dominates the external mass transfer process, roughness could lead to decreased conversion rates. The results of this study clearly indicate that only evaluation of overall conversion rates or mass fluxes can describe the correct biofilm conversion, whereas interpretation of local concentration or flow measurements as such might easily lead to erroneous conclusions.     

Measurement of local diffusion coefficients in biofilms by microinjection and confocal microscopy

A new technique for the determination of local diffusion coefficients in biofilms is described. It is based on the microinjection of fluorescent dyes and quantitative analysis of the subsequent plume formation using confocal laser microscopy. The diffusion coefficients of fluorescein (MW 332), TRITC-IgG (MW 150000) and phycoerythrin (MW 240000) were measured in the cell clusters and interstitial voids of a heterogeneous biofilm. The diffusivities measured in the voids were close to the theoretical values in water. Fluorescein had the same diffusivity in cell clusters, voids, and sterile medium. TRITC-IgG did not diffuse in cell clusters, presumably due to binding to the cell cluster matrix. After treatment of the biofilm with bovine serum albumin, binding capacity decreased and the diffusion coefficient could be measured. The diffusivity of phycoerythrin in cell clusters was impeded by 41%, compared to interstitial voids. From the diffusion data of phycoerythrin it was further calculated that the cell cluster matrix had the characteristics of a gel with 0.6 nm thick fibers and pore diameters of 80 nm. (c) 1997 John Wiley & Sons, Inc.     

A novel method for characterisation of microbial growth kinetics on volatile organic compounds

A novel method for the determination of microbial growth kinetics on hydrophobic volatile organic compounds (VOC) has been developed. A stirred tank reactor was operated as a fed-batch system to which the VOC was continuously fed via the gas phase, assuring a constant VOC concentration in the mineral medium. A flow of air was saturated with the VOC, and then mixed with a further flow of air, to obtain a predetermined VOC concentration. Thus, different VOC concentrations in the mineral medium could be obtained by altering the VOC concentration in the feed gas. The growth kinetics of Xanthobacter autotrophicus GJ10 on 1,2-dichloroethane (DCE) and of Pseudomonas sp. strain JS150 on MonoChloroBenzene (MCB) were assessed using this method. The growth of strain JS150 was strongly inhibited at MCB concentrations higher than 160 mg l⁻¹, and the results were fitted using a piecewise function. The growth kinetics of strain GJ10 were described by the Luong model where maximum growth rate μ_max = 0.12 h⁻¹, substrate saturation constant K _S = 7.8 mg l⁻¹, and maximum substrate concentration S _m (above which growth is completely inhibited) = 1080 mg l⁻¹. Varying nitrogen and oxygen flows enabled the effect of oxygen concentration on the growth kinetics of Pseudomonas JS150 to be determined. Received: 30 November 1998 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

Fluorescence correlation spectroscopy to study diffusion and reaction of bacteriophages inside biofilms

In the natural environment, most of the phages that target bacteria are thought to exist in biofilm ecosystems. The purpose of this study was to gain a clearer understanding of the reactivity of these viral particles when they come into contact with bacteria embedded in biofilms. Experimentally, we quantified lactococcal c2 phage diffusion and reaction through model biofilms using in situ fluorescence correlation spectroscopy with two-photon excitation. Correlation curves for fluorescently labeled c2 phage in nonreacting Stenotrophomonas maltophilia biofilms indicated that extracellular polymeric substances did not provide significant resistance to phage penetration and diffusion, even though penetration and diffusion were sometimes restricted because of the noncontractile tail of the viral particle. Fluctuations in the fluorescence intensity of the labeled phage were detected throughout the thickness of biofilms formed by c2-sensitive and c2-resistant strains of Lactococcus lactis but could never be correlated with time, revealing that the phage was immobile. This finding confirmed that recognition binding receptors for the viral particles were present on the resistant bacterial cell wall. Taken together, our results suggest that biofilms may act as "active" phage reservoirs that can entrap and amplify viral particles and protect them from harsh environments.     

Tracer measurements reveal experimental evidence of biofilm consolidation

The ability to simultaneously measure both biofilm thickness and the mass transfer coefficient of an inert tracer through it provides a powerful method to study biofilm development. In this communication previously published data has been collated to interpret global trends in biofilm structure during the transition towards steady-state. It appears that sudden changes in biofilm structure (directly related to the rate of change of biofilm mass transfer resistance) may occur following transitions in rate of biomass production. These observations are consistent with the concept of consolidation, recently introduced into spatially structured biofilm mathematical models to account for structural realignment of the biofilm under dynamic conditions.