Jennifer L. Nyman

Correlation of Functional Instability and Community Dynamics in Denitrifying Dispersed-Growth Reactors

ABSTRACT Understanding the relationship between microbial community dynamics and functional instability is an important step towards designing reliable biological water treatment systems. In this study, the community dynamics of two dispersed-growth denitrifying reactors were examined during periods of functional stability and instability. In both reactors during the period of functional instability, the effluent chemistry changed over time, with periods of high nitrate concentrations followed by periods of fluctuating nitrite concentrations. Community structure was examined by clone library analysis of the 16S rRNA gene. Community dynamics were investigated with terminal restriction fragment (T-RF) length polymorphism, and the functional diversity represented by T-RFs was assessed through nitrate reduction assays of representative isolates. During the period of functional instability, the community structure changed considerably, and the dynamics correlated significantly with effluent chemistry. The nitrite concentration was significantly correlated with the relative abundances of the nitrate-reducing Delftia- and Achromobacter-like T-RFs. The isolate representing the Acidovorax-like T-RF reduced nitrate directly to nitrogen in batch assays without the accumulation of any intermediates. The Acidovorax-like T-RF relative abundance was significantly negatively correlated with nitrite concentration, indicating that it was associated with good functional performance. The results of this study reveal a clear relationship between community dynamics and functional instability and the importance of diversity among nitrate-reducing populations within a denitrifying community.

Correlation of patterns of denitrification instability in replicated bioreactor communities with shifts in the relative abundance and the denitrification patterns of specific populations

To assess the effects of community structure on the stability of denitrification, six chemostat cultures derived from the same denitrifying community were subjected to step increases in feed nitrate concentration and monitored for evidence that denitrification was either not occurring (indicated by the presence of nitrate) or was incomplete (indicated by the presence of nitrite or nitrous oxide). Functional stability was defined and quantified from the pattern of effluent concentration trends of nitrate and denitrification intermediates. Microbial community structure and dynamics were analyzed by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene. Functional stability varied: one chemostat community lost the ability to reduce all of the influent nitrate; others continued to reduce all of the influent nitrate, but accumulated varying amounts of nitrous oxide. The microbial community structure in two of the chemostats diverged from the others, and variation of functional response among chemostats corresponded with the divergence of community structure. The Acidovorax-like terminal restriction fragment (T-RF) dominated the chemostat that accumulated nitrate, and an Acidovorax-like isolate reduced nitrate directly to dinitrogen gas in batch nitrate reduction assays. In the nitrous oxide-accumulating chemostats, the relative abundance of the Pseudomonas-like T-RF was strongly and significantly correlated with the magnitude of nitrous oxide accumulation, and a Pseudomonas-like isolate accumulated nitrous oxide in batch assays.

Stability in a denitrifying fluidized bed reactor

This study evaluates changes in the microbial community structure and function of a pilot-scale denitrifying fluidized bed reactor during periods of constant operating conditions and periods of perturbation. The perturbations consisted of a shutdown period without feed, two disturbances in which biofilms were mechanically sheared from carrier particles, and a twofold step increase in feed nitrate concentration. In the absence of perturbations, nitrate removal was stable and consistently greater than 99%. The structure and dynamics of the microbial community were studied using cloning and sequencing techniques and terminal restriction fragment length polymorphism (T-RFLP) of the SSU rRNA gene. Under unperturbed operating conditions, stable function was accompanied by high constancy and low variability of community structure with the majority of terminal restriction fragments (T-RFs) appearing throughout operation at consistent relative abundances. Several of the consistently present T-RFs correlated with clone sequences closely related to Acidovorax (98% similarity), Dechloromonas (99% similarity), and Zoogloea (98% similarity), genera recently identified by molecular analyses of similar systems. Significant changes in community structure and function were not observed after the shutdown period. In contrast, following the increase in loading rate and the mechanical disturbances, new T-RFs appeared. After both mechanical disturbances, function and community structure recovered. However, function was much more resilient than community structure. The similarity of response to the mechanical disturbances despite differences in community structure and operating conditions suggests that flexible community structure and potentially the activity of minor members under nonperturbation conditions promotes system recovery.

Uranium (VI) Reduction by Denitrifying Biomass

ABSTRACT Groundwater near the S3 ponds at the US Department of Energys Y-12 site in Oak Ridge, Tennessee, is contaminated by high levels of nitrate (up to 160 mM) and U(VI) (∼0.3 mM). To minimize nitrate inhibition, the authors proposed extraction of contaminated groundwater, nitrate removal in a denitrifying fluidized bed bioreactor (FBR), and return of nitrate-free effluent to the aquifer to stimulate in situ microbial reduction of U(VI). In the presence of carbonate, U(VI) sorption to biomass was negligible, but in its absence, sorption was significant. Biomass reduced U(VI) to U(IV), exhibiting slow first-order removal with respect to U(VI). Addition of electron donor increased rates. Addition of an inhibitor of sulfate reduction (molybdate) slowed the rate and inhibited sulfate reduction. Denitrifying β-Proteobacteria dominated clone libraries of SSU rRNA and dsrA gene sequences. Approximately 10% were low-G+C microorganisms that had 90% to 92% sequence identity with Sporomusa, Acetonema, and Propionispora. The dsrA sequences were dominated by a single clone with ∼80% nucleotide identity to dsrA of Desulfovibrio vulgaris sub sp. oxamicus. The authors conclude that some members of this denitrifyng community reduce uranium, and that sulfate-reducing bacteria likely contribute to this capability.

In-situ determination of field-scale NAPL mass transfer coefficients: Performance, simulation and analysis

Better estimates of non-aqueous phase liquid (NAPL) mass, its persistence into the future, and the potential impact of source reduction are critical needs for determining the optimal path to clean up sites impacted by NAPLs. One impediment to constraining time estimates of source depletion is the uncertainty in the rate of mass transfer between NAPLs and groundwater. In this study, an innovative field test is demonstrated for the purpose of quantifying field-scale NAPL mass transfer coefficients (kl(N)) within a source zone of a fuel-contaminated site. Initial evaluation of the test concept using a numerical model revealed that the aqueous phase concentration response to the injection of clean groundwater within a source zone was a function of NAPL mass transfer. Under rate limited conditions, NAPL dissolution together with the injection flow rate and the radial distance to monitoring points directly controlled time of travel. Concentration responses observed in the field test were consistent with the hypothetical model results allowing field-scale NAPL mass transfer coefficients to be quantified. Site models for groundwater flow and solute transport were systematically calibrated and utilized for data analysis. Results show kl(N) for benzene varied from 0.022 to 0.60d(-1). Variability in results was attributed to a highly heterogeneous horizon consisting of layered media of varying physical properties.

Sulfate Requirement for the Growth of U(VI)-Reducing Bacteria in an Ethanol-Fed Enrichment

ABSTRACT A field experiment at the Oak Ridge Field Research Center has demonstrated the in situ biostimulation of U(VI) reduction with ethanol amendment, but little is known about the stimulated metabolic pathways or composition of the bacterial community mediating the reduction. This work characterized the metabolism and community structure of a sulfate-reducing enrichment developed from sediment from the field site to help address this knowledge gap. Structure was investigated by clone library construction and terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rDNA. The enrichment used ethanol concomitantly with sulfate, producing acetate. Hydrogen accumulated intermittently. The clone library contained sequences related to Clostridia, Desulfovibrio, Bacteroides, and Synergistes species. The enrichment reduced U(VI), and the reduction rate was 0.055 L/mg volatile suspended solids (VSS)/day. The enrichments T-RFLP profile was comprised largely of Desulfovibrio-like fragments, and Desulfovibrio species are known to reduce sulfate and U(VI). A second line of enrichments, inoculated from the sulfate-amended enrichment, was maintained without sulfate. After four transfers of the sulfate-free culture, it was found unable to reduce U(VI). This cultures T-RFLP profile was largely comprised of Clostridia-like fragments, and Clostridia ferment ethanol to acetate. The results indicate a sulfate requirement for the growth of U(VI)-reducing organisms in this community.

Advanced Diagnostic Tools

In the past decade, the advent of innovative diagnostic tools has improved site assessment and remediation practices. This chapter discusses five diagnostic tools that are particularly important for chlorinated solvent source zone remediation: multi-level monitoring systems; rock matrix characterization techniques; mass flux/mass discharge measurements; compound-specific isotope analysis; and molecular biological tools. The discussion includes descriptions of each diagnostic tool, a value of information analysis to help practitioners determine when the tools will be useful and cost effective, and practical recommendations for use of each tool.