Raymond M. Hozalski

Microbial Biofilm Voltammetry: Direct Electrochemical Characterization of Catalytic Electrode-Attached Biofilms†

ABSTRACT While electrochemical characterization of enzymes immobilized on electrodes has become common, there is still a need for reliable quantitative methods for study of electron transfer between living cells and conductive surfaces. This work describes growth of thin (<20 μm) Geobacter sulfurreducens biofilms on polished glassy carbon electrodes, using stirred three-electrode anaerobic bioreactors controlled by potentiostats and nondestructive voltammetry techniques for characterization of viable biofilms. Routine in vivo analysis of electron transfer between bacterial cells and electrodes was performed, providing insight into the main redox-active species participating in electron transfer to electrodes. At low scan rates, cyclic voltammetry revealed catalytic electron transfer between cells and the electrode, similar to what has been observed for pure enzymes attached to electrodes under continuous turnover conditions. Differential pulse voltammetry and electrochemical impedance spectroscopy also revealed features that were consistent with electron transfer being mediated by an adsorbed catalyst. Multiple redox-active species were detected, revealing complexity at the outer surfaces of this bacterium. These techniques provide the basis for cataloging quantifiable, defined electron transfer phenotypes as a function of potential, electrode material, growth phase, and culture conditions and provide a framework for comparisons with other species or communities.

Micro-cantilever method for measuring the tensile strength of biofilms and microbial flocs

Cohesive strength is an important factor in determining the structure and function of biofilm systems, and cohesive strength plays a key role in our ability to remove or control biofilms in engineered systems. A micro-mechanical device has been developed to directly measure the tensile strength of biofilms and other microbial aggregates. An important feature of this method is the combination of a direct measurement of force with particle separations that occur at a scale comparable to that observed for biofilm systems. The force required to separate an aggregate is determined directly from the deflection of cantilevered glass micropipettes with a 20-40-microm diameter. Combined with an estimate of the cross-sectional area of the aggregate at the point of separation this measurement indicates the cohesive strength of the aggregate. Samples of return activated sludge (RAS) and a Pseudomonas aeruginosa biofilm were tested using the device. The measured cohesive strengths of the RAS flocs ranged from 419 to 206,400 N/m(2), while many of the flocs exceeded the range of measurement of the device. Fragments of P. aeruginosa biofilm had cohesive strengths ranging from 395 to 15,640 N/m(2), with a median value of 3020 N/m(2). The median equivalent diameters of the particles detached from the aggregates were 32 microm for RAS and 30 microm for the P. aeruginosa biofilms.

Removal of Natural Organic Matter (NOM) from Drinking Water Supplies by Ozone-Biofiltration

Removal of natural organic matter (NOM) in biofilters can be affected by many factors including NOM characteristics, use of pre-ozonation, water temperature, and biofilter backwashing. Laboratory experiments were performed and a biofilter simulation model was developed for the purpose of evaluating the effects of each of these factors on NOM removal in biofilters. Four sources of NOM were used in this study to represent a broad spectrum of NOM types that may be encountered in water treatment. In batch experiments with raw NOM, the removal of organic carbon by biodegradation was inversely proportional to the UV absorbance (254 nm)-to-TOC ratio and directly proportional to the percentage of low molecular weight material (as determined by ultrafiltration). The extent and rate of total organic carbon (TOC) removal typically increased as ozone dose increased, but the effects were highly dependent on NOM characteristics. NOM with a higher percentage of high molecular weight material experienced the greatest enhancement in biodegradability by ozonation. The performance of laboratory-scale continuous-flow biofilters was not significantly affected by periodic backwashing, because backwashing was unable to remove large amounts of biomass from the filter media. Model simulations confirmed our experimental results and the model was used to further evaluate the effects of temperature and backwashing on biofilter performance.

Effect of NOM and biofilm on the removal of Cryptosporidium parvum oocysts in rapid filters

Laboratory experiments were performed to evaluate the effects of biofilm and natural organic matter (NOM) on removal of Cryptosporidium parvum oocysts from water by filtration. The bench-scale rapid filters consisted of 2.54 cm ID x 30.5 cm polycarbonate plastic columns packed with 0.55 mm spherical glass beads to a depth of 25 cm and a porosity of 40%. Calcium chloride (0.01 M) served as the coagulant in most of the experiments. The oocyst removal efficiency decreased from 51 +/- 6% for a clean bed to 23 +/- 3% for the biofilm-coated bed and to 14 +/- 1% in the presence of 5 ppm of NOM. The oocyst removal for an experiment with a combination of biofilm-coated filter media and NOM was similar to that for the experiment with NOM alone (15 +/- 1%). The zeta potential values for the oocysts pre-equilibrated with NOM were significantly more negative than those obtained for untreated oocysts. This suggests that NOM enhanced the electrostatic repulsion between the oocysts and the negatively charged glass beads. Fortunately, use of alum as coagulant at a dosage sufficient to neutralize the surface charge of the NOM-coated oocysts resulted in a high removal efficiency (73 +/- 6%). Pre-equilibration of the oocysts with NOM also increased the hydrophobicity of the oocysts, but this was deemed to have a negligible effect on deposition onto the glass beads. The results of these experiments suggest that water treatment facilities treating source waters with moderate organic matter concentrations and/or employing biologically active filters have a greater potential for oocyst breakthrough and proper coagulation is critical for effective removal of oocysts in the filters.

Sorption of antibiotics to biofilm.

Using a continuous-flow rotating annular bioreactor, sorption of three selected antibiotics (sulfamethoxazole (SMX), ciprofloxacin (CIP), and erythromycin (ERY)) to bacterial biofilm was investigated. CIP had the greatest biofilm partition coefficient (K(oc) = 92,000 ± 10,000 L/kg) followed by ERY (K(oc) = 6000 ± 1000 L/kg) and then SMX (K(oc) = 4000 ± 1000 L/kg). Antibiotic sorption to biofilm did not correlate with experimentally-determined K(ow) values (CIP: -0.4; ERY: 0.98; SMX: <-0.59 at pH 7), suggesting that hydrophobic interactions do not drive the sorption of these relatively hydrophilic compounds to the biofilm. It appears that speciation (i.e. charge) and molecular size of the antibiotics are important in explaining their sorption to typically negatively charged biofilm. SMX is neutral to negatively charged at circumneutral pH while CIP and ERY are both positively charged. The decreased extent of sorption of ERY relative to CIP is likely due to the larger molecular size of ERY that results in a decreased rate of mass transfer (i.e. diffusion) to and through the biofilm. In conclusion, the results of this research suggest that hydrophobic interactions (predicted by K(ow)) do not control sorption of relatively hydrophilic antibiotics to biofilm and that antibiotic speciation and molecular size are important factors affecting the interactions between antibiotics and biofilm.

Development and testing of a novel microcantilever technique for measuring the cohesive strength of intact biofilms.

Cohesive strength is an important parameter for understanding and modeling the mechanics of biomass detachment from bacterial biofilms. It is challenging to measure the mechanical properties of biofilms, however, because biofilms may desiccate when removed from liquid medium and they are inherently fragile. Poppele and Hozalski (Poppele and Hozalski, 2003, J Microb Methods 55:607–615) presented a microcantilever method for measuring the tensile strength of detached biofilm fragments while submersed in liquid medium. Here we present a modification of the microcantilever method to quantify the strength of intact bacterial biofilms. Initial testing was performed on Pseudomonas aeruginosa biofilms and on Staphylococcus epidermidis biofilms grown in rotating disk reactors. The cohesive strength values were highly variable (i.e., coefficients of variation ranging from 71% to 143%) and ranged from 59 to 18,900 Pa for the P. aeruginosa biofilms and from 61 to 5,840 Pa for the S. epidermidis biofilms. The biofilms also appeared to be isotropic as strength did not vary with angle of testing relative to the direction of applied shear. Strength testing using both the intact and fragment methods was performed on five samples of P. aeruginosa biofilms, and the strength populations were not from the same distribution in three cases. Equivalent diameters for the fragments detached from biofilms during strength testing ranged from 5 to 500 µm, which is within the range of size of biofilm fragments observed in the effluents of lab‐scale and full‐scale bioreactors. The microcantilever is a simple yet powerful tool for measuring the cohesive strength of intact biofilms at a relevant scale. Biotechnol. Bioeng. 2010;105: 924–934.

Passive dissolution of hydrogen gas into groundwater using hollow-fiber membranes

A new hollow-fiber membrane remediation system has recently been developed to passively supply groundwater with dissolved hydrogen (H2) to stimulate the biodegradation of chlorinated solvents. Understanding the mass transfer behavior of membranes under conditions of creeping flow is critical for the design of such systems. Therefore, the objectives of this research were to evaluate the gas transfer behavior of hollow-fiber membranes under conditions typical of groundwater flow and to assess the effect of membrane configuration on gas transfer performance. Membrane gas transfer was evaluated using laboratory-scale glass columns operated at low flow velocities (8.6-12,973 cm/d). H2 was supplied to the inside of the membrane fibers while water flowed on the outside and normal to the fibers (i.e. cross-flow). Membrane configuration (single fiber and fabric) and membrane spacing for the fabric modules did not affect gas transfer performance. Therefore, the results from all of the experiments were combined to obtain the following dimensionless Sherwood number (Sh) correlation expressed as a function of Reynolds number (Re) and Schmidt number (Sc): Sh = 0.824Re(0.39)Sc(0.33) (0.0004<Re<0.6). This correlation is useful for predicting the rate of transfer of any gas from clean membranes to flowing water at low Re. This correlation provides a basis for estimating the membrane surface area requirements for groundwater remediation as illustrated by a simple example.

Assessment of hydrodynamic separators for storm-water treatment

Hydrodynamic separators are proprietary underground devices designed to remove floatable debris e.g., leaves, trash, oil and to remove suspended solids from storm-water runoff by sedimentation. They are designed for storm-water treatment in urban areas to meet tight space constraints. Limited data on the suspended solids removal performance of installed devices are available, and existing data are questionable because of the problems associated with assessment by monitoring. The objectives of our research are to: 1 investigate the feasibility and practicality of field testing to assess the performance of hydrodynamic separators as underground storm-water treatment devices; 2 evaluate the effects of sediment size and storm-water discharge on the performance of six devices from different manufac- turers; and 3 develop a universal approach for predicting the performance of a device for any given application. In the field tests, a controlled and reproducible synthetic storm event containing sediment of a well defined size distribution and concentration was fed to a precleaned device. The captured sediment was then removed, dried, sieved, and weighed. To assess the performance of the devices, suspended sediment removal efficiency was related to a Peclet number, which accounts for two major processes that control performance: 1 settling of particles; and 2 turbulent diffusion or mixing of particles. After analyzing the data, all devices showed similar behavior, therefore, a three-parameter performance function was proposed for all devices. Performance functions were developed from the result of the field tests and parallel testing of two other full-scale devices in the laboratory. The performance functions can be used to determine the efficiency of the tested devices and to improve the selection and sizing of hydrodynamic separators and the assessment of their overall performance after installation.

Evaluation of polyethylene hollow-fiber membranes for hydrogen delivery to support reductive dechlorination in a soil column

Engineered systems are often needed to supply an electron donor, such as hydrogen (H(2)), to the subsurface to stimulate the biological dehalogenation of perchloroethene (PCE) to ethene. A column study was performed to evaluate the ability of gas permeable hollow-fiber membranes to supply H(2) directly to PCE-contaminated groundwater to facilitate bioremediation. Two glass columns were packed with soil obtained from a trichloroethene-contaminated site at Cape Canaveral, Florida, and were fed a minimal medium spiked with PCE (7 microM) for 391 days. The columns were operated in parallel, with one column receiving H(2) via polyethylene hollow-fiber membranes (lumen H(2) pressure of approximately 1atm) and a control column receiving no H(2). PCE was initially dechlorinated at a similar rate and to a similar extent in both columns, likely due to the presence of soil organic matter that was able to support dechlorination. After 265 days of operation, dechlorination performance declined in the control column and the benefits of membrane-supplied H(2) became evident. Although the membrane-supplied H(2) effectively stimulated PCE dechlorination at the end of the experiment (days 359-391), the system was inefficient in that only 5% of the supplied H(2) was used for dechlorination. Most of the remainder was used to support methanogenesis (94%). Despite the dominance of methanogens, nearly complete dechlorination of PCE to ethene was observed in the H(2)-fed column. In addition to the inefficient use of H(2), operational problems included excessive foulant accumulation on the outside of the membrane fibers and water condensation inside the fibers. Use of alternative membrane materials and changes to the operating approach (e.g. pulsing or supplying H(2) at low partial pressures) may help to overcome these problems so that this technology can provide effective and stable remediation of aquifers contaminated with chlorinated ethenes.

Effect of nitrate and sulfate on dechlorination by a mixed hydrogen-fed culture

A novel hollow-fiber membrane remediation technology developed in our laboratory for hydrogen delivery to the subsurface was shown to support the dechlorination of perchloroethene (PCE) to cis-dichloroethene. In previous research, the presence of nitrate or sulfate has been observed to inhibit biological reductive dechlorination. In this study hollow-fiber membranes were used to supply hydrogen to a mixed culture to investigate whether adequate hydrogen could be added to support dechlorination in the presence of alternative electron acceptors. By continuously supplying hydrogen through the membrane, the hydrogen concentrations within the reactor were maintained well above the hydrogen thresholds reported to sustain reductive dechlorination. It was hypothesized that by preventing nitrate and sulfate reducers from decreasing hydrogen concentrations to below the dehalorespirer threshold, the inhibition of PCE dechlorination by nitrate and sulfate might be avoided and dechlorination could be stimulated more effectively. Enough membrane-fed hydrogen was supplied to completely degrade the alternative electron acceptors present and initiate dechlorination. Nevertheless, nitrate and sulfate inhibited dechlorinating activity even when hydrogen was not limiting. This suggests that competition for hydrogen was not responsible for the observed inhibition. Subsequent microcosm experiments demonstrated that the denitrification intermediate nitrous oxide was inhibitory at 13 µM.