Hypochlorous acid-generating electrochemical scaffold eliminates Candida albicans biofilms.

AIMS: Wound infections involving Candida albicans can be challenging to treat because of the fungus' ability to penetrate wound tissue and form biofilms. The goal of this study was to assess the activity of a hypochlorous acid (HOCl)-generating electrochemical scaffold (e-scaffold) against C. albicans biofilms in vitro and on porcine dermal explants (ex vivo). METHODS AND RESULTS: C. albicans biofilms were grown either on acrylic-bottom six-well plates (in vitro) or on skin tissue excised from porcine ears (ex vivo), and the polarized e-scaffold was used to generate a continuous supply of low concentration HOCl near biofilm surfaces. C. albicans biofilms grown in vitro were reduced to undetectable amounts within 24 h of e-scaffold exposure, unlike control biofilms (5·28 ± 0·034 log10  (CFU cm- 2 ); P < 0·0001). C. albicans biofilms grown on porcine dermal explants were also reduced to undetectable amounts in 24 h, unlike control explant biofilms (4·29 ± 0·057 log10 (CFU cm- 2 ); P < 0·0001). There was a decrease in the number of viable mammalian cells (35·6 ± 6·4%) in uninfected porcine dermal explants exposed to continuous HOCl-generating e-scaffolds for 24 h compared to explants exposed to nonpolarized e-scaffolds (not generating HOCl) (P < 0·05). CONCLUSIONS: Our HOCl-generating e-scaffold is a potential antifungal-free strategy to treat C. albicans biofilms in chronic wounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Wound infections caused by C. albicans are difficult to treat due to presence of biofilms in wound beds. Our HOCl producing e-scaffold provides a promising novel approach to treat wound infections caused by C. albicans.

A biofilm microreactor system for simultaneous electrochemical and nuclear magnetic resonance techniques.

Nuclear magnetic resonance (NMR) techniques are ideally suited for the study of biofilms and for probing their microenvironments because these techniques allow for noninvasive interrogation and in situ monitoring with high resolution. By combining NMR with simultaneous electrochemical techniques, it is possible to sustain and study live biofilms respiring on electrodes. Here, we describe a biofilm microreactor system, including a reusable and a disposable reactor, that allows for simultaneous electrochemical and NMR techniques (EC-NMR) at the microscale. Microreactors were designed with custom radio frequency resonator coils, which allowed for NMR measurements of biofilms growing on polarized gold electrodes. For an example application of this system we grew Geobacter sulfurreducens biofilms on electrodes. EC-NMR was used to investigate growth medium flow velocities and depth-resolved acetate concentration inside the biofilm. As a novel contribution we used Monte Carlo error analysis to estimate the standard deviations of the acetate concentration measurements. Overall, we found that the disposable EC-NMR microreactor provided a 9.7 times better signal-to-noise ratio over the reusable reactor. The EC-NMR biofilm microreactor system can ultimately be used to correlate extracellular electron transfer rates with metabolic reactions and explore extracellular electron transfer mechanisms.

Investigating microbial dynamics and potential advantages of anaerobic co-digestion of cheese whey and poultry slaughterhouse wastewaters.

Resource recovery and prevention of environmental pollution are key goals for sustainable development. It is widely reported that agro-industrial activities are responsible for the discharge of billions of liters of wastewater to the environment. Anaerobic digestion of these energy rich agro-industrial wastewaters can simultaneously mitigate environmental pollution and recover embedded energy as methane gas. In this study, an assessment of mono- and co-digestion of cheese whey wastewater (CWW) and poultry slaughterhouse wastewater (PSW) was conducted in 2.25-L lab-scale anaerobic digesters. Treatment combinations evaluated included CWW (R1), PSW (R2), 75:25 CWW:PSW (R3), 25:75 CWW:PSW (R4), and 50:50 CWW:PSW (R5). The digestion efficiencies of the mixed wastewaters were compared to the weighted efficiencies of the corresponding combined mono-digested samples. R4, with a mixture of 25% CWW and 75% PSW, achieved the greatest treatment efficiency. This corresponded with an average biodegradability of 84%, which was greater than for R1 and R2 at 68.5 and 71.9%, respectively. Similarly, R4 produced the highest average cumulative methane value compared to R1 and R2 at 1.22× and 1.39× for similar COD loading, respectively. The modified Gompertz model provided the best fit for the obtained methane production data, with lag time decreasing over progressive treatment cycles. PCoA and heatmap analysis of relative microbial abundances indicated a divergence of microbial communities based on feed type over the treatment cycles. Microbial community analysis showed that genus Petrimonas attained the highest relative abundance (RA) at up to 38.9% in the first two cycles, then subsequently decreased to near 0% for all reactors. Syntrophomonas was highly abundant in PSW reactors, reaching up to 36% RA. Acinetobacter was present mostly in CWW reactors with a RA reaching 56.5%. The methanogenic community was dominated by Methanothrix (84.3-99.9% of archaea). The presence of phosphate and Acinetobacter in CWW feed appeared to reduce the treatment efficiency of associated reactors. Despite Acinetobacter being strictly aerobic, previous and current results indicate its survival under anaerobic conditions, with the storage of phosphate likely playing a key role in its ability to scavenge acetate during the digestion process.

The effect of detachment on biofilm structure and activity: the oscillating pattern of biofilm accumulation.

In our previous papers we have demonstrated that biofilm structure never reaches a steady state in biofilm reactors; in this paper we link this fact to biofilm detachment and to the oscillating pattern of biofilm accumulation. In one respect reactors supporting suspended microbial growth and reactors supporting attached microbial growth (biofilms) are similar: in both the biomass accumulates in the reactor and is disposed of with the effluent. However, while in reactors with suspended microbial growth biomass accumulation and disposal occur simultaneously, in biofilm reactors these two processes are separated in time. Biomass accumulation in biofilm reactors shows a distinct pattern composed of three phases: (1) growth, (2) detachment, (3) regrowth. Despite this distinct pattern of biofilm accumulation observed at the microscale, biofilm reactors do reach a steady state of substrate removal.

Biofilms: their structure, activity, and effect on membrane filtration.

The goal of this presentation is to identify biofouling mechanisms that cause undesirable effects to the membrane separation processes of flux decline and pressure drop. The underlying assumption of this presentation is that biofouling is unavoidable and that the operator cannot eliminate it entirely. This premise justifies research efforts toward understanding the mechanisms by which biofouling affects the membrane processes, rather than expecting that technology can entirely eliminate membrane biofouling in the near future. An improved understanding of biofouling mechanisms may lead to better membrane design, better membrane modules, and better membrane cleaning procedures.

Quantifying biofilm structure using image analysis.

We have developed and implemented methods of extracting morphological features from images of biofilms in order to quantify the characteristics of the inherent heterogeneity. This is a first step towards quantifying the relationship between biofilm heterogeneity and the underlying processes, such as mass-transport dynamics, substrate concentrations, and species variations. We have examined two categories of features, areal, which quantify the relative magnitude of the heterogeneity and textural, which quantify the microscale structure of the heterogeneous elements. The feature set is not exhaustive and has been restricted to two-dimensional images to this point. Included in this paper are the methods used to extract the structural information and the algorithms used to quantify the data. The features discussed are porosity, fractal dimension, diffusional length, angular second moment, inverse difference moment and textural entropy. We have found that some features are better predictors of biofilm behavior than others and we discuss possible future directions for research in this area.

Growing reproducible biofilms with respect to structure and viable cell counts.

We have developed a new method of growing 4-day-old biofilms that are reproducible, with respect to viable cell number and biofilm structure. To demonstrate the utility of the method, we grew biofilms composed of Pseudomonas aeruginosa (ATCC#700829), P. fluorescens (ATCC#700830) and Klebsiella pneumoniae (ATCC#700831), 18 times in flat-plate reactors under well-defined conditions of: flow rate, nutrient concentration, temperature, inoculum and growth rate. The resulting 4-day-old biofilms were approximately 200-300 microm thick and exhibited a high degree of reproducibility. The number of viable cells that accumulated per unit surface area and the biofilm areal porosity were reproduced within 10% error. We have also quantified other parameters characterizing biofilm structure using biofilm-imaging techniques: fractal dimension, textural entropy and diffusion distance as auxiliary parameters characterizing the reproducibility of biofilm accumulation. As a result of analysis, we have introduced a new parameter to better quantify and characterize the number of viable cells in biofilms, "specific number of viable cells" (SNVC). This parameter is the viable cell number normalized with respect to the surface area covered by the biofilm and with respect to the biomass of the biofilm. This new descriptor represents the dynamics of biofilm accumulation better than the traditionally used colony-forming unit (CFU) per surface area covered by the biofilm because it accounts not only for the surface coverage but also for the biofilm thickness.

Evaluation of biofilm image thresholding methods.

To evaluate biomass distribution in heterogeneous biofilms from their microscope images, it is often necessary to perform image thresholding by converting the gray-scale images to binary images consisting of a foreground of biomass material and a background of interstitial space. The selection of the gray-scale intensity used for thresholding is arbitrary but under the control of the operator, which may produce unacceptable levels of variability among operators. The quality of numerical information extracted from the images is diminished by such variability, and it is desirable to find a method that improves the reproducibility of thresholding operations. Automatic methods of thresholding provide this reproducibility, but often at the expense of accuracy, as they consistently set thresholds that differ significantly from what human operators would choose. The performance of five automatic image thresholding algorithms was tested in this study: (1) local entropy; (2) joint entropy; (3) relative entropy; (4) Renyi's entropy; and (5) iterative selection. Only the iterative selection method was satisfactory in that it was consistently setting the threshold level near that set manually. The extraction of feature information from biofilm images benefits from automatic thresholding and can be extended to other fields, such as medical imaging.

Biofilm monitoring: a perfect solution in search of a problem.

The main problem with monitoring biofilms is data interpretation. Biofilm heterogeneity causes monitored parameters to vary from location to location in the same biofilm, and it is difficult to assess to what extent these variations are caused by biofilm heterogeneity and to what extent they reflect other properties of the biofilm. We have used the concept of discretized biofilms, which is an integrated system of biofilm monitoring and data interpretation, to assess the effect of biofilm heterogeneity on biofilm activity. Using this approach we have estimated that a heterogeneous biofilm can be ten times more active, in terms of glucose consumption rate, than a homogeneous biofilm of the same thickness but with uniformly distributed density.

Reproducibility of biofilm processes and the meaning of steady state in biofilm reactors.

The need for reproducing biofilm processes is undisputable - the quality of biofilm research depends on this reproducibility. However, as many biofilm researchers know, long-term biofilm processes are notoriously difficult to reproduce. To avoid problems related to biofilm reproducibility two strategies are used: (1) to study very young biofilms that have accumulated for a few hours to a few days only, and (2) to run biofilm experiments only once. The first approach trades reproducibility for relevance because natural biofilms are usually older, often much older than a few days. This approach can be applied to answer questions relevant to initial events of biofilm formation but not questions relevant to long-term biofilm accumulation. The second approach conceals the problem of biofilm reproducibility. To assure reproducibility of biofilm processes, we methodically followed a procedure for growing biofilms in terms of microbial makeup, media composition, temperature, surface preparation, etc. Despite all this effort the reproducibility of our results for long term growth is unimpressive. Consequently, the question had to be asked: Are biofilm processes reproducible? The experiments described in this paper address this question. Biofilms grown in two identical and identically operated biofilm reactors had comparable structure only until the first sloughing event. After that, biofilms had different patterns of accumulation.

DIFFUSION IN BIOFILMS RESPIRING ON ELECTRODES.

The goal of this study was to measure spatially and temporally resolved effective diffusion coefficients (D(e)) in biofilms respiring on electrodes. Two model electrochemically active biofilms, Geobacter sulfurreducens PCA and Shewanella oneidensis MR-1, were investigated. A novel nuclear magnetic resonance microimaging perfusion probe capable of simultaneous electrochemical and pulsed-field gradient nuclear magnetic resonance (PFG-NMR) techniques was used. PFG-NMR allowed noninvasive, nondestructive, high spatial resolution in situ D(e) measurements in living biofilms respiring on electrodes. The electrodes were polarized so that they would act as the sole terminal electron acceptor for microbial metabolism. We present our results as both two-dimensional D(e) heat maps and surface-averaged relative effective diffusion coefficient (D(rs)) depth profiles. We found that 1) D(rs) decreases with depth in G. sulfurreducens biofilms, following a sigmoid shape; 2) D(rs) at a given location decreases with G. sulfurreducens biofilm age; 3) average D(e) and D(rs) profiles in G. sulfurreducens biofilms are lower than those in S. oneidensis biofilms-the G. sulfurreducens biofilms studied here were on average 10 times denser than the S. oneidensis biofilms; and 4) halting the respiration of a G. sulfurreducens biofilm decreases the D(e) values. Density, reflected by D(e), plays a major role in the extracellular electron transfer strategies of electrochemically active biofilms.

METABOLIC SPATIAL VARIABILITY IN ELECTRODE-RESPIRING GEOBACTER SULFURREDUCENS BIOFILMS.

In this study, we quantified electron transfer rates, depth profiles of electron donor, and biofilm structure of Geobacter sulfurreducens biofilms using an electrochemical-nuclear magnetic resonance microimaging biofilm reactor. Our goal was to determine whether electron donor limitations existed in electron transfer processes of electrode-respiring G. sulfurreducens biofilms. Cells near the top of the biofilms consumed acetate and were metabolically active; however, acetate concentration decreased to below detection within the top 100 microns of the biofilms. Additionally, porosity in the biofilms fell below 10% near the electrode surface, exacerbating exclusion of acetate from the lower regions. The dense biofilm matrix in the acetate-depleted zone acted as an electrical conduit passing electrons generated at the top of the biofilm to the electrode. To verify the distribution of cell metabolic activity, we used uranium as a redox-active probe for localizing electron transfer activity and X-ray absorption spectroscopy to determine the uranium oxidation state. Cells near the top reduced UVI more actively than the cells near the base. High-resolution transmission electron microscopy images showed intact, healthy cells near the top and plasmolyzed cells near the base. Contrary to models proposed in the literature, which hypothesize that cells nearest the electrode surface are the most metabolically active because of a lower electron transfer resistance, our results suggest that electrical resistance through the biofilm does not restrict long-range electron transfer. Cells far from the electrode can respire across metabolically inactive cells, taking advantage of their extracellular infrastructure produced during the initial biofilm formation.

Prediction of average biofilm density and performance of a spherical bioparticle under substrate inhibition.

In this article, a model was proposed to predict the average performance and biofilm density of a spherical bioparticle under substrate inhibition in a fluidized bed system. The average biofilm density and substrate consumption rates were predicted for a definite biofilm thickness and limiting substrate concentrations. A diffusion and reaction model was developed over the bioparticle with biofilm-density dependent effective diffusion coefficients for maximum substrate consumption theory. This theory predicts the optimum density of a biofilm to yield a maximum substrate consumption rate within the biofilm, developed for the first time with this study and experimentally verified. A good correlation was observed between the model prediction and experimental results for biofilm density and substrate consumption rates.

Fiber-optic microsensors to measure backscattered light intensity in biofilms.

We have constructed tapered fiber-optic microsensors with a tip diameter of less than 10 microm to measure profiles of backscattered light in biofilms, which are thin layers of micro-organisms firmly attached to surfaces. The observed response agrees well with local effective diffusivity microelectrode measurements, with R(2) > 0.85. A strong relation between signal intensity and wavelength has been observed at 670 and 1320 nm. These sensors have the potential to replace local effective diffusivity microelectrodes for true in situ biofilm measurements.