Investigating the potential for microbially induced carbonate precipitation to treat mine waste.

In this study, the feasibility of promoting microbially induced carbonate precipitation (MICP) in mine waste piles by using an environmental bacterial enrichment is explored, with goals to reduce metals and acid leaching. MICP has been explored for remediation applications and stabilization of mine waste. Here, we utilize a native bacterial enrichment to promote MICP on seven mine waste samples with variability in acid production and extent of toxic metal leaching. During fifteen applications of MICP solutions and bacteria on waste rock in bench-scale columns, calcium carbonate formed on grain surfaces within all waste samples, though microscopy revealed uneven distribution of CaCO3 coating. The effluent from acid-producing wastes increased in pH during MICP treatment. MICP performance was evaluated with humidity cell and synthetic precipitation leaching procedure (SPLP) tests. Leaching tests revealed reductions in Cd, Pb and Zn concentrations in leachate of all but one sample, mixed results for Cu, and As increasing in all but one leachate sample after treatment. MICP technology has potential for coating mine waste and reducing release of acid and some metals. This study provides a laboratory assessment of MICP feasibility for stabilizing mine waste in situ and mitigating release of toxic metals into the environment.

Controlling the Distribution of Microbially Precipitated Calcium Carbonate in Radial Flow Environments.

Bacterially driven reactions such as ureolysis can induce calcium carbonate precipitation, a well-studied process called microbially induced calcium carbonate precipitation (MICP). MICP is of interest in subsurface applications such as sealing leaks around wells. For effective field deployment, it is important to study MICP under radial flow conditions, which are relevant to near-well environments. In this study, a laboratory-scale radial flow reactor of 23 cm diameter, with a 1 mm glass bead monolayer serving as a porous medium, was used to investigate the effects of fluid flow rates and calcium concentrations on the mass and distribution of MICP by the ureolytic bacterium Sporosarcina pasteurii. Experiments were performed at hydraulic residence times of 14, 7, and 3.5 min and calcium to urea molar ratios of 0.5:1, 1:1, and 2:1. The total amount of CaCO3 precipitated in the reactor increased with increasing residence time and with decreasing Ca2+ to urea molar ratios. Increased bacterial attachment and increased CaCO3 precipitation were observed with distance from the center inlet of the reactor in all experiments. More uniform calcium distribution was achieved at lower flow rates. The relationship between reaction and transport rate (i.e., the Damköhler number) is identified as a useful parameter for the prediction of MICP in radial flow environments.

Evaluation of Biofilm Induced Urinary Infection Stone Formation in a Novel Laboratory Model System.

PURPOSE: Infection stones, which comprise approximately 15% of all urinary tract stones, are induced by infection with urease-positive pathogens. The bacteria in the stone matrix present significant treatment impediments compared to metabolic kidney stones. While much is known about how urinary composition regulates metabolic stone formation, there is a general lack of knowledge of which urinary factors regulate the rate of infection stone formation. Unfortunately more in-depth research into infection stones is limited by the lack of suitable models for real-time study of bacterial biofilm formation and stone formation under varying conditions. MATERIALS AND METHODS: We developed an in vitro model to study infection stone formation. The model closely represents the processes that occur in vivo, including the observed migration of ureolytic bacteria (our culture of Proteus mirabilis) from the bladder to the kidneys, followed by biofilm and stone formation in the kidney. We used scanning electron and confocal laser microscopy, x-ray diffraction, biological counts and dissolved chemical analyses to evaluate the model system. RESULTS: Crystals that formed in the system resembled clinically removed struvite stones in structure and composition. Results showed that the degree of ureolysis required to significantly change urine pH was minimal, bacterial communities inhabited the ureter, and upstream colonization and struvite formation required lag time. CONCLUSIONS: These results have implications for the detection and treatment of struvite stones. Currently this model is being used to study specific urinary factors that regulate struvite formation to identify treatment options, which combined with antibiotics would improve treatment of these stones and decrease recurrence.

Impact of Mineral Precipitation on Flow and Mixing in Porous Media Determined by Microcomputed Tomography and MRI.

Precipitation reactions influence transport properties in porous media and can be coupled to advective and dispersive transport. For example, in subsurface environments, mixing of groundwater and injected solutions can induce mineral supersaturation of constituents and drive precipitation reactions. Magnetic resonance imaging (MRI) and microcomputed tomography (µ-CT) were employed as complementary techniques to evaluate advection, dispersion, and formation of precipitate in a 3D porous media flow cell. Two parallel fluids were flowed concentrically through packed glass beads under two relative flow rates with Na2CO3 and CaCl2 in the inner and outer fluids, respectively. CaCO3 became supersaturated and formed a precipitate at the mixing interface between the two solutions. Spatial maps of changing local velocity fields and dispersion in the flow cell were generated from MRI, while high resolution µ-CT imaging visualized the precipitate formed in the porous media. Formation of a precipitate minimized dispersive and advective transport between the two fluids and the shape of the precipitation front was influenced by the relative flow rates. This work demonstrates that the combined use of MRI and µ-CT can be highly complementary in the study of reactive transport processes in porous media.

Kinetic parameter estimation in N. europaea biofilms using a 2-D reactive transport model.

Biofilms of the ammonia oxidizing bacterium Nitrosomonas europaea were cultivated to study microbial processes associated with ammonia oxidation in pure culture. We explored the hypothesis that the kinetic parameters of ammonia oxidation in N. europaea biofilms were in the range of those determined with batch suspended cells. Oxygen and pH microelectrodes were used to measure dissolved oxygen (DO) concentrations and pH above and inside biofilms and reactive transport modeling was performed to simulate the measured DO and pH profiles. A two dimensional (2-D) model was used to simulate advection parallel to the biofilm surface and diffusion through the overlying fluid while reaction and diffusion were simulated in the biofilm. Three experimental studies of microsensor measurements were performed with biofilms: i) NH3 concentrations near the Ksn value of 40 µM determined in suspended cell tests ii) Limited buffering capacity which resulted in a pH gradient within the biofilms and iii) NH3 concentrations well below the Ksn value. Very good fits to the DO concentration profiles both in the fluid above and in the biofilms were achieved using the 2-D model. The modeling study revealed that the half-saturation coefficient for NH3 in N. europaea biofilms was close to the value measured in suspended cells. However, the third study of biofilms with low availability of NH3 deviated from the model prediction. The model also predicted shifts in the DO profiles and the gradient in pH that resulted for the case of limited buffering capacity. The results illustrate the importance of incorporating both key transport and chemical processes in a biofilm reactive transport model.

Inhibition of phenol on the rates of ammonia oxidation by Nitrosomonas europaea grown under batch, continuous fed, and biofilm conditions.

Ammonia oxidation by Nitrosomonas europaea, an ammonia oxidizing bacterium prevalent in wastewater treatment, is inhibited in the presence of phenol, due to interaction of the phenol with the ammonia monooxygenase enzyme. Suspended cells of N. europaea were cultured in batch reactors and continuous flow reactors at dilution rates of 0.01-0.2 d(-1). The rate of ammonia oxidation in the continuous cultures correlated to the dilution rate in the reactor. The batch and continuous cultures were exposed to 20 µM phenol and ammonia oxidation activity was measured by specific oxygen uptake rates (SOURs). Inhibition of NH3 oxidation by 20 µM phenol ranged from a 77% reduction of SOUR observed with suspended cells harvested during exponential growth, to 26% in biofilms. The extent of inhibition was correlated with ammonia oxidation rates in both suspended and biofilm cells, with greater percent inhibition observed with higher initial rates of NH3 oxidation. In biofilm grown cells, an increase in activity and phenol inhibition were both observed upon dispersing the biofilm cells into fresh, liquid medium. Under higher oxygen tension, an increase in the NO2(-) production of the biofilms was observed and biofilms were more susceptible to phenol inhibition. Dissolved oxygen microsensor measurements showed oxygen limited conditions existed in the biofilms. The ammonia oxidation rate was much lower in biofilms, which were less inhibited during phenol exposure. The results clearly indicate in both suspended and attached cells of N. europaea that a higher extent of phenol inhibition is positively correlated with a higher rate of NH3 oxidation (enzyme turnover).

Bacterially induced calcium carbonate precipitation and strontium coprecipitation in a porous media flow system.

Strontium-90 is a principal radionuclide contaminant in the subsurface at several Department of Energy sites in the Western U.S., causing a threat to groundwater quality in areas such as Hanford, WA. In this work, we used laboratory-scale porous media flow cells to examine a potential remediation strategy employing coprecipitation of strontium in carbonate minerals. CaCO(3) precipitation and strontium coprecipitation were induced via ureolysis by Sporosarcina pasteurii in two-dimensional porous media reactors. An injection strategy using pulsed injection of calcium mineralization medium was tested against a continuous injection strategy. The pulsed injection strategy involved periods of lowered calcite saturation index combined with short high fluid velocity flow periods of calcium mineralization medium followed by stagnation (no-flow) periods to promote homogeneous CaCO(3) precipitation. By alternating the addition of mineralization and growth media the pulsed strategy promoted CaCO(3) precipitation while sustaining the ureolytic culture over time. Both injection strategies achieved ureolysis with subsequent CaCO(3) precipitation and strontium coprecipitation. The pulsed injection strategy precipitated 71-85% of calcium and 59% of strontium, while the continuous injection was less efficient and precipitated 61% of calcium and 56% of strontium. Over the 60 day operation of the pulsed reactors, ureolysis was continually observed, suggesting that the balance between growth and precipitation phases allowed for continued cell viability. Our results support the pulsed injection strategy as a viable option for ureolysis-induced strontium coprecipitation because it may reduce the likelihood of injection well accumulation caused by localized mineral plugging while Sr coprecipitation efficiency is maintained in field-scale applications.

Investigating Nitrosomonas europaea stress biomarkers in batch, continuous culture, and biofilm reactors.

The understanding of nitrification inhibition in ammonia oxidizing bacteria (AOB) by priority pollutants and emerging contaminants is critical in managing the nitrogen cycle to preserve current water supplies, one of the National Academy of Engineers Grand Challenges in Engineering for the twenty-first century. Nitrosomonas europaea is an excellent model AOB for nitrification inhibition experimentation due to its well-defined NH(3) metabolism and the availability of a wide range of physiological and transcriptional tools that can characterize the mechanism of nitrification inhibition and probe N. europaea's response to the inhibitor. This chapter is a compilation of the physiological and transcriptional methods that have been used to characterize nitrification inhibition of N. europaea under a wide variety of growth conditions including batch, continuously cultured, and in biofilms. The protocols presented here can be applied to other AOB, and may be readily adapted for other autotrophic bacteria (e.g., nitrite oxidizing bacteria).

Inhibition and gene expression of Nitrosomonas europaea biofilms exposed to phenol and toluene.

Pure culture biofilms of the ammonia-oxidizing bacterium Nitrosomonas europaea were grown in a Drip Flow Biofilm Reactor and exposed to the aromatic hydrocarbons phenol and toluene. Ammonia oxidation rates, as measured by nitrite production in the biofilms, were inhibited 50% when exposed to 56 µM phenol or 100 µM toluene, while 50% inhibition of suspended cells occurred at 8 µM phenol or 20 µM toluene. Biofilm-grown cells dispersed into liquid medium and immediately exposed to phenol or toluene experienced similar inhibition levels as batch grown cells, indicating that mass transfer may be a factor in N. europaea biofilm resistance. Whole genome microarray analysis of gene expression was used to detect genes up-regulated in biofilms during toluene and phenol exposure. Two genes, a putative pirin protein (NE1545) and a putative inner membrane protein (NE1546) were up-regulated during phenol exposure, but no genes were up-regulated during toluene exposure. Using qRT-PCR, up-regulation of NE1545 was detected in biofilms and suspended cells exposed to a range of phenol concentrations and levels of inhibition. In the biofilms, NE1545 expression was up-regulated an average of 13-fold over the range of phenol concentrations tested, and was essentially independent of phenol concentration. However, the expression of NE1545 in suspended cells increased from 20-fold at 7 µM phenol up to 80-fold at 30 µM phenol. This study demonstrates that biofilms of N. europaea are more resistant than suspended cells to inhibition of ammonia oxidation by phenol and toluene, even though the global transcriptional responses to the inhibitors do not differ in N. europaea between the suspended and attached growth states.