Kela P. Weber

One-dimensional metric for tracking bacterial community divergence using sole carbon source utilization patterns.

Community level physiological profiling (CLPP) has become a popular method to characterize and track changes in heterotrophic bacterial communities. Although the CLPP method is a straight forward laboratory protocol which yields large amounts of functional information, the amount of data obtained can become overwhelming and often requires some type of multivariate analysis method for ordination and interpretation. Multivariate analysis can be challenging and requires a significant statistics background along with an understanding of the inferences and biases each multivariate analysis method incurs. This paper presents and evaluates a new approach to analyzing sole carbon source utilization data. A method is described which provides a one-dimensional metric derived from standard CLPP data (BIOLOG EcoPlate data). The one-dimensional community metric was derived using normalized Euclidean distances and shifts in the carbon source utilization patterns. The one-dimensional community metric did not provide all of the information of classical approaches such as principle component analysis (PCA) or guild grouping analysis; however, it was found to be more easily implemented and interpreted when analyzing the plate data. Validation of this approach is demonstrated using data acquired to track the divergence of bacterial communities in wetland mesocosm systems after an experimentally controlled disturbance. If the objective is to investigate community shifts over time the one-dimensional community divergence metric can be a useful tool.

Characterization of nZVI Mobility in a Field Scale Test

Nanoscale zerovalent iron (nZVI) particles were injected into a contaminated sandy subsurface area in Sarnia, Ontario. The nZVI was synthesized on site, creating a slurry of 1 g/L nanoparticles using the chemical precipitation method with sodium borohydride (NaBH4) as the reductant in the presence of 0.8% wt. sodium carboxymethylcellulose (CMC) polymer to form a stable suspension. Individual nZVI particles formed during synthesis had a transmission electron microscopy (TEM) quantified particle size of 86.0 nm and dynamic light scattering (DLS) quantified hydrodynamic diameter for the CMC and nZVI of 624.8 nm. The nZVI was delivered to the subsurface via gravity injection. Peak normalized total Fe breakthrough of 71% was observed 1m from the injection well and remained above 50% for the 24 h injection period. Samples collected from a monitoring well 1 m from the injection contained nanoparticles with TEM-measured particle diameter of 80.2 nm and hydrodynamic diameter of 562.9 nm. No morphological changes were discernible between the injected nanoparticles and nanoparticles recovered from the monitoring well. Energy dispersive X-ray spectroscopy (EDS) was used to confirm the elemental composition of the iron nanoparticles sampled from the downstream monitoring well, verifying the successful transport of nZVI particles. This study suggests that CMC stabilized nZVI can be transported at least 1 m to the contaminated source zone at significant Fe(0) concentrations for reaction with target contaminants.

Community-Level Physiological Profiling

Community-level physiological profiling (CLPP) is a technique which offers an easily applied protocol yielding information regarding mixed microbial community function and functional adaptations over space and time. Different communities can be compared and classified based on sole carbon source utilization patterns (CSUPs) gathered using BIOLOG microplates. One of the most challenging aspects associated with the CLPP method is in the data analysis. This chapter describes the relatively simple CLPP laboratory protocol and provides a detailed description of different data analysis techniques.

Contributions of Abiotic and Biotic Dechlorination Following Carboxymethyl Cellulose Stabilized Nanoscale Zero Valent Iron Injection

A pilot scale injection of nanoscale zerovalent iron (nZVI) stabilized with carboxymethyl cellulose (CMC) was performed at an active field site contaminated with a range of chlorinated volatile organic compounds (cVOC). The cVOC concentrations and microbial populations were monitored at the site before and after nZVI injection. The remedial injection successfully reduced parent compound concentrations on site. A period of abiotic degradation was followed by a period of enhanced biotic degradation. Results suggest that the nZVI/CMC injection created conditions that stimulated the native populations of organohalide-respiring microorganisms. The abundance of Dehalococcoides spp. immediately following the nZVI/CMC injection increased by 1 order of magnitude throughout the nZVI/CMC affected area relative to preinjection abundance. Distinctly higher cVOC degradation occurred as a result of the nZVI/CMC injection over a 3 week evaluation period when compared to control wells. This suggests that both abiotic and biotic degradation occurred following injection.

Long-Term Field Study of Microbial Community and Dechlorinating Activity Following Carboxymethyl Cellulose-Stabilized Nanoscale Zero-Valent Iron Injection

Nanoscale zerovalent iron (nZVI) is an emerging technology for the remediation of contaminated sites. However, there are concerns related to the impact of nZVI on in situ microbial communities. In this study, the microbial community composition at a contaminated site was monitored over two years following the injection of nZVI stabilized with carboxymethyl cellulose (nZVI-CMC). Enhanced dechlorination of chlorinated ethenes to nontoxic ethene was observed long after the expected nZVI oxidation. The abundance of Dehalococcoides (Dhc) and vinyl chloride reductase (vcrA) genes, monitored using qPCR, increased by over an order of magnitude in nZVI-CMC-impacted wells. The entire microbial community was tracked using 16S rRNA gene amplicon pyrosequencing. Following nZVI-CMC injection, a clear shift in microbial community was observed, with most notable increases in the dechlorinating genera Dehalococcoides and Dehalogenimonas. This study suggests that coupled abiotic degradation (i.e., from reaction with nZVI) and biotic degradation fueled by CMC led to the long-term degradation of chlorinated ethenes at this field site. Furthermore, nZVI-CMC addition stimulated dehalogenator growth (e.g., Dehalococcoides) and biotic degradation of chlorinated ethenes.

Method for the detachment of culturable bacteria from wetland gravel

The study of bacterial communities in microbially-mediated water treatment systems is becoming increasingly popular. Aquatic bacterial communities are often found in fixed-film environments, residing within a matrix of extracellular polymeric substances commonly referred to as a biofilm. A method for detaching the biofilm is required to either enumerate or characterize these bacterial communities. There are a variety of detachment methods including scraping, swabbing, shaking, sonication, blending, and digestion. The objective of this work was to develop an agitation-based protocol for detachment of culturable bacterial communities from the biofilm surrounding pea gravel from constructed wetland mesocosms. Three different protocol factors were systematically investigated using a triplicated 2(3) factorial design to determine the most effective detachment protocol. Factors studied included: the use of either tap water or phosphate buffer as the shaking/detachment solution; the use of either manual-shaking at room temperature or mechanical shaking at 30 degrees C; and the presence or absence of an enzyme cocktail consisting of lipase, beta-galactosidase and alpha-glucosidase. The resulting suspensions were evaluated for organics, inorganics, culturable bacteria, community level physiological profile (CLPP) and several BIOLOG ECO plate substrate related diversity indices. Using these metrics, the most effective shaking/detachment protocol was identified as mechanical shaking for 3h at 30 degrees C using a phosphate buffer with an enzyme cocktail.

Comparison of the catabolic activity and catabolic profiles of rhizospheric, gravel-associated and interstitial microbial communities in treatment wetlands

Microbial communities play a critical role in degrading organic contaminants in treatment wetlands; however, an understanding of the different roles played by rhizospheric, gravel-associated and interstitial microbial communities is deficient due to a lack of data directly comparing these microbial communities. Community level physiological profiling (CLPP) was used to compare the catabolic capabilities of rhizospheric, gravel-associated and interstitial microbial communities in vertical-flow planted and unplanted wetland mesocosms. Wetland mesocosms were decommissioned to gather microbial community samples associated with the roots and gravel bed media taken from the top (10 cm depth), middle (30 cm depth) and bottom (60 cm depth). The catabolic capabilities of the rhizospheric microbial communities were seen to be much greater than those of the gravel-associated communities. A decrease in catabolic capability was seen with increasing depth, suggesting that communities near the surface play a larger role in the degradation of carbon-based compounds. A general difference in catabolic profiles based on plant presence/absence was observed for the interstitial water and all gravel-associated samples at all depths, suggesting that the presence of roots within part of the mesocosm not only has a localized effect on the attached microbial population, but also on gravel-associated microbial communities throughout the mesocosms.

Functionality of microbial communities in constructed wetlands used for pesticide remediation: Influence of system design and sampling strategy

The objective of this study was to compare the microbial community metabolic function from both unsaturated and saturated constructed wetland mesocosms (CWs) when treating the pesticide tebuconazole. The comparison was performed for both interstitial water and substrate biofilm by community level physiological profiling (CLPP) via BIOLOG™ EcoPlates. For each CW design (saturated or unsaturated), six mesocosms were established including one unplanted and five planted individually with either Juncus effusus, Typha latifolia, Berula erecta, Phragmites australis or Iris pseudacorus. Microbial activity and metabolic richness of interstitial water from unsaturated CWs were significantly lower than that from saturated CWs. However, in general, the opposite result was observed for biofilm samples. Wetland plants promoted significantly higher biofilm microbial activity and metabolic richness than unplanted CWs in both CW designs. Differences in the microbial community functional profiles between plant species were only found for saturated CWs. Biofilm microbial metabolic richness was generally statistically higher than that of interstitial water in both unsaturated (1.4-24 times higher) and saturated (1.2-1.7 times higher) CWs. Carbon source (guild) utilization patterns were generally different between interstitial water and biofilm samples. Functionality of the biofilm microbial community was positively correlated to the removal of all pollutants (TN, NH4+-N, TP, TOC and tebuconazole) for both unsaturated and saturated CWs, suggesting the biofilm plays a more important role in pollutant removal than the interstitial water microbial community. Thus, merely observing the interstitial water microbial communities may underestimate the role of the microbial community in CW performance. Interestingly, the ability for the biofilm microbial community to utilize amino acids and amines/amides was positively correlated with tebuconazole removal in all system types.

nZVI injection into variably saturated soils: Field and modeling study

Nano-scale zero valent iron (nZVI) has been used at a number of contaminated sites over the last decade. At most of these sites, significant decreases in contaminant concentrations have resulted from the application of nZVI. However, limited work has been completed investigating nZVI field-scale mobility. In this study, a field test was combined with numerical modeling to examine nZVI reactivity along with transport properties in variably saturated soils. The field test consisted of 142L of carboxymethyle cellulose (CMC) stabilized monometallic nZVI synthesized onsite and injected into a variably saturated zone. Periodic groundwater samples were collected from the injection well, as well as, from two monitoring wells to analyze for chlorinated solvents and other geochemistry indicators. This study showed that CMC stabilized monometallic nZVI was able to decrease tricholorethene (TCE) concentrations in groundwater by more than 99% from the historical TCE concentrations. A three dimensional, three phase, finite difference numerical simulator, (CompSim) was used to further investigate nZVI and polymer transport at the variably saturated site. The model was able to accurately predict the field observed head data without parameter fitting. In addition, the numerical simulator estimated the mass of nZVI delivered to the saturated and unsaturated zones and distinguished the nZVI phase (i.e. aqueous or attached). The simulation results showed that the injected slurry migrated radially outward from the injection well, and therefore nZVI transport was governed by injection velocity and viscosity of the injected solution. A suite of sensitivity analyses was performed to investigate the impact of different injection scenarios (e.g. different volume and injection rate) on nZVI migration. Simulation results showed that injection of a higher nZVI volume delivered more iron particles at a given distance; however, the travel distance was not proportional to the increase in volume. Moreover, simulation results showed that using a 1D transport equation to simulate nZVI migration in the subsurface may overestimate the travel distance. This is because the 1D transport equation assumes a constant velocity while pore water velocity radially decreases from the well during injection. This study suggests that on-site synthesized nZVI particles are mobile in the subsurface and that a numerical simulator can be a valuable tool for optimal design of nZVI field applications.

Nitrification cessation and recovery in an aerated saturated vertical subsurface flow treatment wetland: Field studies and microscale biofilm modeling.

In aerated treatment wetlands, oxygen availability is not a limiting factor in sustaining a high level of nitrification in wastewater treatment. In the case of an air blower failure, nitrification would cease, potentially causing adverse effects to the nitrifying bacteria. A field trial was completed investigating nitrification loss when aeration is switched off, and the system recovery rate after the aeration is switched back on. Loss of dissolved oxygen was observed to be more rapid than loss of nitrification. Nitrate was observed in the effluent long after the aeration was switched off (48h+). A complementary modeling study predicted nitrate diffusion out of biofilm over a 48h period. After two weeks of no aeration in the established system, nitrification recovered within two days, whereas nitrification establishment in a new system was previously observed to require 20-45days. These results suggest that once established resident nitrifying microbial communities are quite robust.