Heidi L. Gough

Linkage of high rates of sulfate reduction in Yellowstone hot springs to unique sequence types in the dissimilatory sulfate respiration pathway.

ABSTRACT Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89°C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.

Microbial community structures in anoxic freshwater lake sediment along a metal contamination gradient

Contamination, such as by heavy metals, has frequently been implicated in altering microbial community structure. However, this association has not been extensively studied for anaerobic communities, or in freshwater lake sediments. We investigated microbial community structure in the metal-contaminated anoxic sediments of a eutrophic lake that were impacted over the course of 80 years by nearby zinc-smelting activities. Microbial community structure was inferred for bacterial, archaeal and eukaryotic populations by evaluating terminal restriction fragment length polymorphism (TRFLP) patterns in near-surface sediments collected in triplicate from five areas of the lake that had differing levels of metal contamination. The majority of the fragments in the bacterial and eukaryotic profiles showed no evidence of variation in association with metal contamination levels, and diversity revealed by these profiles remained consistent even as metal concentrations varied from 3000 to 27 000 mg kg−1 total Zn, 0.125 to 11.2 μ pore water Zn and 0.023 to 5.40 μM pore water As. Although most archaeal fragments also showed no evidence of variation, the prevalence of a fragment associated with mesophilic Crenarchaeota showed significant positive correlation with total Zn concentrations. This Crenarchaeota fragment dominated the archaeal TRFLP profiles, representing between 35% and 79% of the total measured peak areas. Lake DePue 16S rRNA gene sequences corresponding to this TRFLP fragment clustered with anaerobic and soil mesophilic Crenarchaeota sequences. Although Crenarchaeota have been associated with metal-contaminated groundwater and soils, this is a first report (to our knowledge) documenting potential increased prevalence of Crenarchaeota associated with elevated levels of metal contamination.

Functional gene array–based analysis of microbial communities in heavy metals-contaminated lake sediments

Lake DePue (IL, USA) has been contaminated for > 80 years by an adjacent Zn-smelting facility. Previous work indicated that sulfate reduction increased and biomass declined as pore-water metal concentrations increased, while 16S rRNA gene profiles remained relatively stable. To better understand this phenomenon, the sediment microbial community structure and functional potential were investigated using a functional gene microarray (GeoChip) targeting > 10,000 functional genes. Nonmetric multidimensional scaling and clustering analyses showed that the overall community structure was similar across all sites based on the relative abundance of all detected genes, but some individual gene categories did show differences. A subset of sulfate reduction genes (dsr) and the most relevant metal resistance genes were more abundant than other categories and were highly correlated with metal contamination. The most significant correlations were between pore-water metal concentrations and dsr, with Zn, Cd, and Mn as the most predictive for the presence of dsr. These results suggest that metal contamination influences sediment microbial community structure and function by increasing the abundance of relevant metal-resistant and sulfate-reducing populations. These populations therefore appear to contribute significantly to the resistance and stability of the microbial communities throughout the gradient of metal contamination in Lake DePue.

Monitoring the dynamics of syntrophic β-oxidizing bacteria during anaerobic degradation of oleic acid by quantitative PCR

The ecophysiology of long-chain fatty acid-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic β-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic β-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid β-oxidization potential of anaerobic digester communities.

Identification of Putative Genes Involved in Bisphenol A Degradation Using Differential Protein Abundance Analysis of Sphingobium sp. BiD32

Discharge of the endocrine disrupting compound bisphenol A (BPA) with wastewater treatment plant (WWTP) effluents into surface waters results in deleterious effects on aquatic life. Sphingobium sp. BiD32 was previously isolated from activated sludge based on its ability to degrade BPA. This study investigated BPA metabolism by Sphingobium sp. BiD32 using label-free quantitative proteomics. The genome of Sphingobium sp. BiD32 was sequenced to provide a species-specific platform for optimal protein identification. The bacterial proteomes of Sphingobium sp. BiD32 in the presence and absence of BPA were identified and quantified. A total of 2155 proteins were identified; 1174 of these proteins were quantified, and 184 of these proteins had a statistically significant change in abundance in response to the presence/absence of BPA (p ≤ 0.05). Proteins encoded by genes previously identified to be responsible for protocatechuate degradation were upregulated in the presence of BPA. The analysis of the metabolites from BPA degradation by Sphingobium sp. BiD32 detected a hydroxylated metabolite. A novel p-hydroxybenzoate hydroxylase enzyme detected by proteomics was implicated in the metabolic pathway associated with the detected metabolite. This enzyme is hypothesized to be involved in BPA degradation by Sphingobium sp. BiD32, and may serve as a future genetic marker for BPA degradation.

Genomic, Proteomic, and Metabolite Characterization of Gemfibrozil-Degrading Organism Bacillus sp. GeD10.

Gemfibrozil is a widely used hypolipidemic and triglyceride lowering drug. Excess of the drug is excreted and discharged into the environment primarily via wastewater treatment plant effluents. Bacillus sp. GeD10, a gemfibrozil-degrader, was previously isolated from activated sludge. It is the first identified bacterium capable of degrading gemfibrozil. Gemfibrozil degradation by Bacillus sp. GeD10 was here studied through genome sequencing, quantitative proteomics and metabolite analysis. From the bacterial proteome of Bacillus sp. GeD10 1974 proteins were quantified, of which 284 proteins were found to be overabundant by more than 2-fold (FDR corrected p-value ≤0.032, fold change (log2) ≥ 1) in response to gemfibrozil exposure. Metabolomic analysis identified two hydroxylated intermediates as well as a glucuronidated hydroxyl-metabolite of gemfibrozil. Overall, gemfibrozil exposure in Bacillus sp. GeD10 increased the abundance of several enzymes potentially involved in gemfibrozil degradation as well as resulted in the production of several gemfibrozil metabolites. The potential catabolic pathway/modification included ring-hydroxylation preparing the substrate for subsequent ring cleavage by a meta-cleaving enzyme. The identified genes may allow for monitoring of potential gemfibrozil-degrading organisms in situ and increase the understanding of microbial processing of trace level contaminants. This study represents the first omics study on a gemfibrozil-degrading bacterium.

Kinetics modeling predicts bioaugmentation with Sphingomonad cultures as a viable technology for enhanced pharmaceutical and personal care products removal during wastewater treatment

Pharmaceutical and personal care products (PPCPs) discharged with wastewater treatment effluents are a surface water quality concern. PPCPs are partially removed during wastewater treatment and biological transformation is an important removal mechanism. To investigate the potential for enhanced PPCP removal using bioaugmentation, bacteria were previously isolated from activated sludge capable of degrading PPCPs to ng/L concentrations. This study examined the degradation kinetics of triclosan and bisphenol A by five of these bacteria, both in pure culture and when augmented to activated sludge. Sorption coefficients were determined to account for the influence of partitioning during bioremoval. When the bacteria were added to activated sludge, degradation increased. Experimentally determined kinetic parameters were used to model a full-scale continuous treatment process, showing that low biomass could achieve reduced effluent PPCP concentrations. These results demonstrated that bioaugmentation may improve PPCP removal using established wastewater infrastructure under conditions of high solids partitioning.

Earthworms (Eisenia fetida) demonstrate potential for use in soil bioremediation by increasing the degradation rates of heavy crude oil hydrocarbons

Crude oil contamination widely impacts soil as a result of release during oil and gas exploration and production activities. The success of bioremediation methods to meet remediation goals often depends on the composition of the crude oil, the soil, and microbial community. Earthworms may enhance bioremediation by mixing and aerating the soil, and exposing soil microorganisms to conditions in the earthworm gut that lead to increased activity. In this study, the common composting earthworm Eisenia fetida was tested for utility to improve remediation of oil-impacted soil. E. fetida survival in soil contaminated with two distinct crude oils was tested in an artificial (lab-mixed) sandy loam soil, and survival compared to that in the clean soil. Crude oil with a high fraction of light-weight hydrocarbons was more toxic to earthworms than the crude oil with a high proportion of heavy polyaromatic and aliphatic hydrocarbons. The heavier crude oil was added to soil to create a 30,000mg/kg crude oil impacted soil, and degradation in the presence of added earthworms and feed, feed alone, or no additions was monitored over time and compared. Earthworm feed was spread on top to test effectiveness of no mixing. TPH degradation rate for the earthworm treatments was ~90mg/day slowing by 200days to ~20mg/day, producing two phases of degradation. With feed alone, the rate was ~40mg/day, with signs of slowing after 500days. Both treatments reached the same end point concentrations, and exhibited faster degradation of aliphatic hydrocarbons C21, decreased. During these experiments, soils were moderately toxic during the first three months, then earthworms survived well, were active and reproduced with petroleum hydrocarbons present. This study demonstrated that earthworms accelerate bioremediation of crude oil in soils, including the degradation of the heaviest polyaromatic fractions.

Influence of bioselector processes on 17α-ethinylestradiol biodegradation in activated sludge wastewater treatment systems.

The removal of the potent endocrine-disrupting estrogen hormone, 17α-ethinylestradiol (EE2), in municipal wastewater treatment plant (WWTP) activated sludge (AS) processes can occur through biodegradation by heterotrophic bacteria growing on other organic wastewater substrates. Different kinetic and metabolic substrate utilization conditions created with AS bioselector processes can affect the heterotrophic population composition in AS. The primary goal of this research was to determine if these changes also affect specific EE2 biodegradation kinetics. A series of experiments were conducted with parallel bench-scale AS reactors treating municipal wastewater with estrogens at 100-300 ng/L concentrations to evaluate the effect of bioselector designs on pseudo first-order EE2 biodegradation kinetics normalized to mixed liquor volatile suspended solids (VSS). Kinetic rate coefficient (kb) values for EE2 biodegradation ranged from 5.0 to 18.9 L/g VSS/d at temperatures of 18 °C to 24 °C. EE2 kb values for aerobic biomass growth at low initial food to mass ratio feeding conditions (F/Mf) were 1.4 to 2.2 times greater than that from growth at high initial F/Mf. Anoxic/aerobic and anaerobic/aerobic metabolic bioselector reactors achieving biological nutrient removal had similar EE2 kb values, which were lower than that in aerobic AS reactors with biomass growth at low initial F/Mf. These results provide evidence that population selection with growth at low organic substrate concentrations can lead to improved EE2 biodegradation kinetics in AS treatment.

Characterization of Persistent Virus-Like Particles in Two Acetate-Fed Methanogenic Reactors

The objective of this study was to characterize the morphology, size-distribution, concentration and genome size of virus-like particles (VLPs) in two acetate-fed Methanosaeta-dominated reactors to better understand the possible correlation between viruses and archaeal hosts. The study reactors were dominated by a single genus of acetoclastic methanogen, Methanosaeta, which was present at 6 to 13 times higher than the combined bacterial populations consisting of Proteobacteria, Firmicutes, and Bacteroidetes. Epifluorescent microscopy showed VLPs concentration of 7.1 ± 1.5×107 VLPs/ml and 8.4 ± 4.3×107 VLPs/ml in the two laboratory reactors. Observations of no detectable import of VLPs with the reactor feed combined long operational time since the last inocula were introduced suggests that the VLP populations were actively propagating in the reactors. Transmission electron microscopy images showed VLPs with morphology consistent with Siphoviridae in both reactors, and VLPs with morphologies consistent with Myoviridae in one of the reactors. The morphology, size-distribution and genome size of VLPs were distinct between reactors suggesting that unique viral populations inhabited each reactor, though the hosts of these VLPs remain unclear.