Shear-induced detachment of biofilms from hollow fiber silicone membranes.

A suite of techniques was utilized to evaluate the correlation between biofilm physiology, fluid-induced shear stress, and detachment in hollow fiber membrane aerated bioreactors. Two monoculture species biofilms were grown on silicone fibers in a hollow fiber membrane aerated bioreactors (HfMBR) to assess detachment under laminar fluid flow conditions. Both physiology (biofilm thickness and roughness) and nutrient mass transport data indicated the presence of a steady state mature biofilm after 3 weeks of development. Surface shear stress proved to be an important parameter for predicting passive detachment for the two biofilms. The average shear stress at the surface of Nitrosomonas europaea biofilms (54.5 ± 3.2 mPa) was approximately 20% higher than for Pseudomonas aeruginosa biofilms (45.8 ± 7.7 mPa), resulting in higher biomass detachment. No significant difference in shear stress was measured between immature and mature biofilms of the same species. There was a significant difference in detached biomass for immature vs. mature biofilms in both species. However, there was no difference in detachment rate between the two species.

A self referencing platinum nanoparticle decorated enzyme-based microbiosensor for real time measurement of physiological glucose transport.

Glucose is the central molecule in many biochemical pathways, and numerous approaches have been developed for fabricating micro biosensors designed to measure glucose concentration in/near cells and/or tissues. An inherent problem for microsensors used in physiological studies is a low signal-to-noise ratio, which is further complicated by concentration drift due to the metabolic activity of cells. A microsensor technique designed to filter extraneous electrical noise and provide direct quantification of active membrane transport is known as self-referencing. Self-referencing involves oscillation of a single microsensor via computer-controlled stepper motors within a stable gradient formed near cells/tissues (i.e., within the concentration boundary layer). The non-invasive technique provides direct measurement of trans-membrane (or trans-tissue) analyte flux. A glucose micro biosensor was fabricated using deposition of nanomaterials (platinum black, multiwalled carbon nanotubes, Nafion) and glucose oxidase on a platinum/iridium microelectrode. The highly sensitive/selective biosensor was used in the self-referencing modality for cell/tissue physiological transport studies. Detailed analysis of signal drift/noise filtering via phase sensitive detection (including a post-measurement analytical technique) are provided. Using this highly sensitive technique, physiological glucose uptake is demonstrated in a wide range of metabolic and pharmacological studies. Use of this technique is demonstrated for cancer cell physiology, bioenergetics, diabetes, and microbial biofilm physiology. This robust and versatile biosensor technique will provide much insight into biological transport in biomedical, environmental, and agricultural research applications.

Membrane-aerated biofilm proton and oxygen flux during chemical toxin exposure.

Bioreactors containing sessile bacteria (biofilms) grown on hollow fiber membranes have been used for treatment of many wastestreams. Real time operational control of bioreactor performance requires detailed knowledge of the relationship between bulk liquid water quality and physiological transport at the biofilm-liquid interface. Although large data sets exist describing membrane-aerated bioreactor effluent quality, very little real time data is available characterizing boundary layer transport under physiological conditions. A noninvasive, microsensor technique was used to quantify real time (≈1.5 s) changes in oxygen and proton flux for mature Nitrosomonas europaea and Pseudomonas aeruginosa biofilms in membrane-aerated bioreactors following exposure to environmental toxins. Stress response was characterized during exposure to toxins with known mode of action (chlorocarbonyl cyanide phenyl-hydrazone and potassium cyanide), and four environmental toxins (rotenone, 2,4-dinitrophenol, cadmium chloride, and pentachlorophenol). Exposure to sublethal concentrations of all environmental toxins caused significant increases in O(2) and/or H(+) flux (depending on the mode of action). These real time microscale signatures (i.e., fingerprints) of O(2) and H(+) flux can be coupled with bulk liquid analysis to improve our understanding of physiology in counter-diffusion biofilms found within membrane aerated bioreactors; leading to enhanced monitoring/modeling strategies for bioreactor control.

Non-invasive self-referencing electrochemical sensors for quantifying real-time biofilm analyte flux.

Current techniques for characterizing biofilm physiology lack the signal filtering capability required for quantifying signals associated with real time biologically active transport. Though a great deal was learned from previous investigations, no results have been reported on the characterization of in vivo, real time biofilm flux using non-invasive (non-destructive) techniques. This article introduces the self-referencing technique for applications in biofilm physiology. Self-referencing is a non-invasive sensing modality which is capable of sensing changes in biologically active analyte flux as small as 10 fmol cm(-2) s(-1). Studies directly characterizing flux, as opposed to concentration, have the advantage of quantifying real time changes in biologically active transport which are otherwise lost to background noise. The use of this modality for characterizing biofilm physiology is validated with a reversible enzyme inhibition study. The experiment used self-referencing potentiometric sensors for quantifying real time ammonium and nitrite flux. Amperometric and optical sensing methods, though not presented herein, are also powerful sensing tools which benefit from operation in self-referencing mode. Reversible ammonia monooxygenase inhibition by a copper chelator (thiourea), and subsequent relief by excess copper addition was successfully demonstrated using self-referencing ion-selective microelectrodes for a mature Nitrosomonas europaea biofilm.

Dissipation of PAHs in saturated, dredged sediments: a field trial.

Sediments dredged from navigable rivers often contain elevated concentrations of recalcitrant, potentially toxic organic compounds such as polychlorinated biphenyls (PCBs) and polyaromatic hydrocarbons (PAHs). The presence of these compounds often requires that the sediments be stored in fully contained disposal facilities. A 3-year field study was conducted at the Jones Island disposal facility in Milwaukee, Wisconsin, to compare bioremediation of PAHs in contaminated dredged sediments in the absence of plants to phytoremediation with Salix nigra (black willow) (SX61), Spartina pectinata (prairie cord grass), Carex aquatalis (lake sedge), Lolium multiflorum (annual rye), and Scirpus fluviatilis (bulrush). Nine PAHs were detected initially in the sediments. Over the 3-year experiment, acenaphthene dissipation ranged from 94% to 100%, whereas anthracene, benzo[a]pyrene and indo[1,2,3-cd]pyrene generally had modest decreases in concentration (0-30% decrease). The remaining five PAHs ranged in degree of disappearance from 23% to 82%. Planted treatments did not enhance PAH dissipation relative to those without plants, but treatments with high biomass yield and high transpiration plant species had significantly less removal of PAHs than unplanted controls. Significant, negative correlations between nitrogen removal and decreases in PAH concentration suggest that competition for nutrients between plants and microorganisms may have impeded the microbial degradation of PAHs in the rhizosphere of the more rapidly growing plant species.

Influence of organic acids on the transport of heavy metals in soil.

Vegetation historically has been an important part of reclamation of sites contaminated with metals, whether the objective was to stabilize the metals or remove them through phytoremediation. Understanding the impact of organic acids typically found in the rhizosphere would contribute to our knowledge of the impact of plants in contaminated environments. Heavy metal transport in soils in the presence of simple organic acids was assessed in two laboratory studies. In the first study, thin layer chromatography (TLC) was used to investigate Zn, Cd, and Pb movement in a sandy loam soil as affected by soluble organic acids in the rhizosphere. Many of these organic acids enhanced heavy metal movement. For organic acid concentrations of 10mM, citric acid had the highest R(f) values (frontal distance moved by metal divided by frontal distance moved by the solution) for Zn, followed by malic, tartaric, fumaric, and glutaric acids. Citric acid also has the highest R(f) value for Cd movement followed by fumaric acid. Citric acid and tartaric acid enhanced Pb transport to the greatest degree. For most organic acids studied, R(f) values followed the trend Zn>Cd>Pb. Citric acid (10mM) increased R(f) values of Zn and Cd by approximately three times relative to water. In the second study, small soil columns were used to test the impact of simple organic acids on Zn, Cd, and Pb leaching in soils. Citric acid greatly enhanced Zn and Cd movement in soils but had little influence on Pb movement. The Zn and Cd in the effluents from columns treated with 10mM citric acid attained influent metal concentrations by the end of the experiment, but effluent metal concentrations were much less than influent concentrations for citrate <10mM. Exchangeable Zn in the soil columns was about 40% of total Zn, and approximately 80% total Cd was in exchangeable form. Nearly all of the Pb retained by the soil columns was exchangeable.

Biodegradation of polyalcohol ethoxylate by a wastewater microbial consortium.

Polyalcohol ethoxylate (PAE), an anionic surfactant, is the primary component in most laundry and dish wash detergents and is therefore highly loaded in domestic wastewater. Its biodegradation results in the formation of several metabolites and the fate of these metabolites through wastewater treatment plants, graywater recycling processes, and in the environment must be clearly understood. Biodegradation pathways for PAE were investigated in this project with a municipal wastewater microbial consortium. A microtiter-based oxygen sensor system was utilized to determine the preferential use of potential biodegradation products. Results show that while polyethylene glycols (PEGs) were readily degraded by PAE acclimated microorganisms, most of the carboxylic acids tested were not degraded. Biodegradation of PEGs suggests that hydrophobe-hydrophile scission was the dominant pathway for PAE biodegradation in this wastewater community. Ethylene glycol (EG) and diethylene glycol (DEG) were not utilized by microbial populations capable of degrading higher molecular weight EGs. It is possible that EG and DEG may accumulate. The microtiter-based oxygen sensor system was successfully utilized to elucidate information on PAE biodegradation pathways and could be applied to study biodegradation pathways for other important contaminants.

Phytoremediation of polychlorinated biphenyl (PCB)-contaminated sediment: a greenhouse feasibility study.

Contaminated sediments dredged from navigable waterways often are placed in confined disposal facilities to prevent further spread of the pollutants. Reducing contaminants to acceptable levels would allow for disposal of the sediments and further dredging activity. A greenhouse study was conducted to evaluate plant treatments and the addition of an organic amendment to decrease the concentration of PCB congeners found in Arochlor 1260. Sediment treated with the amendment and either low transpiring plants or no plants had the greatest removal of the PCB congeners. High-transpiring plants apparently prevented the highly reducing conditions required for reductive dechlorination of highly chlorinated PCBs. Most likely, the amendment provided labile carbon that initiated the reducing conditions needed for dechlorination. The sediment moisture content and moisture-related plant parameters were significant predictors of the PCB loss. Carex aquatalis and Spartina pectinata are predicted to be the most effective plant treatments for phytoremediation of PCBs.

Heavy metal leaching from mine tailings as affected by organic amendments.

A column experiment was conducted to investigate Zn, Cd, and Pb leaching from mine tailings as affected by the addition of organic amendments. Composted yard waste, composted cattle manure, and cattle manure aged for one month increased heavy metal leaching from mine tailings when compared to an unamended control. Aged cattle manure and composted cattle manure significantly increased Zn concentration in the leachate. The maximum Zn concentration in leachate from the manure-amended treatments was as high as 3.7 mg/L, whereas Zn concentrations from the control were less than 0.7 mg/L. All organic amendments increased Cd leachate concentrations. The presence of aged cattle manure greatly increased Pb concentrations in the leachate from less than 10 microg/L for the control treatment to higher than 60 microg/L. Lead concentration in leachate was positively correlated with inorganic carbon, total organic carbon, total carbon and bicarbonate. Although organic amendments increased Zn, Cd, and Pb leaching when compared with the control treatment, Zn concentrations were lower than the 5 mg/L secondary drinking water standard, and Pb concentrations were only minimally higher than the 15 microg/L drinking water standard. Cadmium concentrations from manure treatments exceeded the 5 microg/L drinking water standard but only during the first 15 days. Organic amendments may encourage establishment of vegetation in mining areas that may minimize heavy metal contamination through runoff and erosion. However, increased risk due to heavy metal leaching in the presence of organic amendments should be carefully considered.

Impact of microbial/plant interactions on the transformation of polycyclic aromatic hydrocarbons in rhizosphere of Festuca arundinacea.

The promotion of polycyclic aromatic hydrocarbon (PAH) degradation was demonstrated in the rhizosphere of Festuca arundinacea with Pseudomonas fluorescens. P. fluorescens 5RL more significantly interacted with salicylate and dextrose in the agar containing tall fescue than agar without plant roots. Although the presence of tall fescue did not promote catabolic enzyme induction in the absence of salicylate, an increase in dioxygenase activity relative to no plant controls implies that this plant may enhance the degradation of PAHs or facilitate the genotypes that are capable of transforming PAH in the rhizosphere.

Biosolids-amended soils: Part I. Effect of biosolids application on soil quality and ecotoxicity.

Investigations of potential risk from biosolids generally indicate that land application does not threaten human or ecosystem health, but questions continue to arise concerning the environmental effects of this practice. This research project was initiated to evaluate ecotoxicity resulting from the amendment of soils with biosolids from municipal wastewater treatment plants. Toxicity was evaluated using standard tests, including earthworm mortality, growth, and reproduction; seedling germination and root elongation; microbial respiration; and nematode mortality and reproduction. Nineteen municipal wastewater treatment plants were identified to participate in an initial screening of toxicity, and five were chosen for a more detailed evaluation. In addition, two soils with historically high applications of high-metal biosolids were evaluated. Contaminants examined were zinc, copper, nickel, chromium, arsenic, cadmium, lead, and coplanar polychlorinated biphenyls (PCBs). Single applications had no effect on soil metal concentrations. Coplanar PCBs were not detectable in any of the soils or biosolids. All target organisms were sensitive to reference toxicants. Limited toxicity was observed in a small number of the amended soils, but no patterns emerged. Approximately one-half of the negative effects of biosolids on bioindicators could be attributed to routine properties, such as slight depression of pH and/or elevated salinity. None of the accumulated metal concentrations were excessive, and most would not be considered elevated. These observations suggest that current regulations for application of biosolids to soils are providing adequate ecosystem protection.

Biosolids-amended soils: Part II. Chemical lability as a measure of contaminant bioaccessability.

Biosolids recycling by amending agricultural soils has increased significantly over the last few decades. The presence of contaminants in small, bioavailable quantities has generated concerns about health threats resulting from accumulation of potential toxins in the food chain. In this study, land application of biosolids was evaluated for environmental risk. Chemical lability tests for metals were used for the test soils and included analyses for water soluble, exchangeable, and metals extractable by the physiologically based extraction test. Chemical extractions detected slight increases in labile metal concentrations for many of the treated soils, particularly those receiving long-term applications of 5 years or more. Significantly higher metal concentrations were observed in the soils that had been exposed to biosolids before the U.S. Environmental Protection Agency (Washington, D.C.) 503 Rule (U.S. EPA, 2004) was implemented.

Lead stabilization by phosphate amendments in soil impacted by paint residue.

The addition of phosphate was evaluated for contaminant stabilization in soils impacted by lead paint residue. Soils sampled from 15 highway bridge sites in Indiana were screened based on residual lead concentrations from paint contamination. Two appropriate bridge sites were identified in Tippecanoe County, Indiana. Soluble phosphate was added to the soil at a mole ratio of 3:1 P:Pb. The efficacy of phosphate treatment was evaluated by a physiologically based extraction test (PBET), uptake of lead by sunflowers, and leaching of lead from soil columns. Sunflowers were established on both field sites, and the mean Pb concentration in the above-ground biomass indicated that the rate of uptake was similar to plants growing in uncontaminated soil. The second bioavailability assessment was the physiologically based extraction test, designed to evaluate heavy metal availability during ingestion. After 1 year at both sites, the addition of phosphate significantly reduced the concentrations of lead extracted by PBET, indicating that the lead in the amended soils had lower bioavailability than in the unamended soils. In the column study, the contaminated soil produced the highest mass of leached Pb, and the addition of P reduced the mass of Pb in the leachate to similar levels found in the uncontaminated soil. Overall, the addition of soluble phosphate to these soils appears to be an effective approach for immobilizing Pb and reducing the associated bio-accessibility.

Characteristics of blast furnace slag leachate produced under reduced and oxidized conditions.

A laboratory study was conducted to determine the environmental conditions necessary to reproduce leachates observed emerging from blast furnace slag acting as the foundation of highways in northwest Indiana. The leachates in the field are often highly alkaline with a pungent sulfur odor, a distinct green or milky-white in color, and sulfate concentrations exceeding 2,000 mg/L. Slag was equilibrated in the laboratory under both oxidized and anoxic environments and at various slag:water ratios. Constant anoxic conditions were required to produce to green colors in the slag, but high sulfate concentrations were observed only when the suspensions were fully oxidized. Leachate from the study site appears to form as a result of a series of complex chemical reactions including fluctuating oxidized and reduced conditions.

Ecotoxicity of pentachlorophenol in contaminated soil as affected by soil type.

Four uncontaminated soils were chosen with a wide range of pH, organic carbon, and clay content to allow us to determine the properties that were most influential on pentachlorophenol (PCP) toxicity. The soils were contaminated in the laboratory at concentrations of 50 and 100 mg/kg and target organisms were exposed to the contaminated soil. Germination and emergence of lettuce seedlings was found to be dependent upon PCP concentration and soil type, and responses were highly correlated to extractable concentrations. Earthworms were sensitive to PCP, regardless of soil properties, and mortality was observed in most samples at the 100 mg/kg concentration. Toxic responses by the worms were not strongly related to soil properties or extractable concentrations. The importance of soil chemical and physical properties on toxicity and bioavailability depends upon the target organism. In the case of lettuce seedlings, PCP is acquired through the aqueous phase; therefore, the chemical interaction between PCP and soil controls toxicity. Since earthworms ingest soil and potentially can change the chemical environment of exposure, the impact of soil properties on PCP toxicity is less apparent.

Leaching and reduction of chromium in soil as affected by soil organic content and plants.

The oxidation state of chromium in contaminated soils is an important indicator of toxicity and potential mobility. Chromium in the hexavalent state is highly toxic and soluble, whereas the trivalent state is much less toxic and relatively insoluble. A laboratory study investigated the impact of growing plants and supplemental organic matter on chromium transport in soil. Plants alone had no appreciable effect on the chromium oxidation state in soil. Soil columns with higher organic content were associated with lower ratios of chromate:total chromium than the columns with lower organic matter. Analyses of column leachate, plant biomass, and soil indicate that more chromium leaching occurred in the vegetated, low organic columns. Retention of Cr in the soils was correlated to the Cr(III) content. Plant uptake of chromium accounted for less than 1% of the chromium removed from the soil. Overall, the addition of organic matter had the strongest influence on chromium mobility.

The influence of organic ligands on the retention of lead in soil.

Organic acids are commonly produced and exuded by plant roots and soil microorganisms. Some of these organic compounds are effective chelating agents and have the potential to enhance metal mobility. The effect of citrate and salicylate on the leaching of lead in soil was investigated in a laboratory experiment. In short-term batch experiments, adsorption of lead to soil was slightly enhanced with increasing salicylate concentration (500-5000 microM) but decreased significantly in the presence of citrate. These observations suggested that citrate may enhance Pb leaching, but this was not observed in the column study. Soluble Pb in the presence and absence citrate or salicylate (up to 5000 microM) was added to soil columns at a moderate flow rate, but no Pb was observed to emerge from the soil in any of the soil columns. Rapid biodegradation of citrate in soil eliminated potential complexing ability. Breakthrough of Pb from soil was noted only when using small columns at high flow rates (>20 pore volumes per day). Under these conditions of physical and chemical non-equilibrium, citrate was not degraded and significantly enhanced Pb mobility. As in the batch adsorption experiments, the presence of salicylate reduced Pb leaching. Considering the extreme conditions required to induce Pb leaching, it is likely that Pb will remain relatively immobile in soil even in the presence of a strong complexing agent such as citrate.

Phytoextraction of Zn and Cu from sewage sludge and impact on agronomic characteristics.

The presence of elevated concentrations of heavy metals limits the usage of sewage sludge as a fertilizer and soil amendment. Experiments were carried out to examine the extent to which seven plant species phytoextracted Zn and Cu from dewatered sludge. The hyperaccumulators Thlaspi caerulescens and Sedum alfredii showed the greatest removal of Zn, while shoots and tubers of two species of Alocasia showed the greatest Cu removal. Cultivation of plants in the sludge resulted in significant decreases in total Zn and changes in the partitioning of Zn between soil pools. However, Cu levels were largely unchanged and remained associated predominantly with the organic matter pool. Agronomic characteristics of the sludge material, such as pH, organic matter content, and nitrogen, phosphorus, and potassium concentrations, did not change significantly during the four-month growth period, indicating that subsequent crops could be sustained by this material. These results suggest that Zn can be phytoextracted from sludge material, provided the rate of metal uptake exceeds the rate of mobilization to the exchangeable fraction. Since there was no appreciable accumulation of Zn and Cu in seeds of Zea mays in this study, some tissues from sludge-grown plants could potentially be used as animal fodder.

The effect of plants on the degradation and toxicity of petroleum contaminants in soil: a field assessment.

A field project located at the US Naval Base at Port Hueneme, California was designed to evaluate changes in contaminant concentrations and toxicity during phytoremediation. Vegetated plots were established in petroleum (diesel and heavy oil) contaminated soil and were evaluated over a two-year period. Plant species were chosen based on initial germination studies and included native California grasses. The toxicity of the impacted soil in vegetated and unvegetated plots was evaluated using Microtox, earthworm, and seed germination assays. The reduction of toxicity was affected more by contaminant aging than the establishment of plants. However, total petroleum hydrocarbon concentrations were lower by the end of the study in the vegetated plots when compared to the unvegetated soil. Although phytoremediation is an effective approach for cleaning-up of petroleum contaminated soil, a long-term management plan is required for significant reductions in contaminant concentrations.

Degradation of crude oil in the rhizosphere of Sorghum bicolor.

Dissipation of petroleum contaminants in the rhizosphere is likely the result of enhanced microbial degradation. Plant roots may encourage rhizosphere microbial activity through exudation of nutrients and by providing channels for increased water flow and gas diffusion. Phytoremediation of crude oil in soil was examined in this study using carefully selected plant species monitored over specific plant growth stages. Four sorghum (Sorghum bicolor L.) genotypes with differing root characteristics and levels of exudation were established in a sandy loam soil contaminated with 2700 mg crude oil/kg soil. Soils were sampled at three stages of plant growth: five leaf, flowering, and maturity. All vegetated treatments were associated with higher remediation efficiency, resulting in significantly lower total petroleum hydrocarbon concentrations than unvegetated controls. A relationship between root exudation and bioremediation efficiency was not apparent for these genotypes, although the presence of all sorghum genotypes resulted in significant removal of crude oil from the impacted soil.