James G. Moberly

Molecular Studies on the Microbial Diversity Associated with Mining-Impacted Coeur d’Alene River Sediments

The prokaryotic diversity associated with highly metal-contaminated sediment samples collected from the Coeur d’Alene River (CdAR) was investigated using a cultivation-independent approach. Bacterial community structure was studied by constructing an RNA polymerase beta subunit (rpoB) gene library. Phylogenetic analysis revealed that 75.8% of the rpoB clones were associated with β-Proteobacteria while the remaining 24.2% were with γ-Proteobacteria. All phylotypes showed close similarity to previously reported cultivable lineages from metal or organic contaminant-rich environments. In an archaeal 16S rRNA gene library, 70% of the clones were affiliated to Crenarchaeota, while 30% belonged to Euryarchaeota. Most of the Euryarchaeota sequences were related to acetoclastic lineages belonging to Methanosarcinales. A single phylotype within the Euryarchaeota showed no association with cultivable euryarchaeotal lineages and might represent novel taxon. Diversity indices demonstrated greater diversity of Bacteria compared to Archaea in CdAR sediments. Sediment characterization by the X-ray fluorescence spectroscopy revealed high amount of toxic metals. To our knowledge, this is the first culture-independent survey on the prokaryotic diversity present in mining-impacted sediments of CdAR.

Influence of pH and Inorganic Phosphate on Toxicity of Zinc to Arthrobacter sp. Isolated from Heavy-Metal-Contaminated Sediments

Because of its high solubility over a wide range of pH conditions, zinc is found in many natural and human-impacted systems. Zinc speciation is critical in assessing zinc toxicity to microorganisms because it varies considerably with pH and is dependent on other aqueous constituents. Combined results of thermodynamic modeling, statistical analysis, and batch culture studies using Arthrobacter sp. JM018 suggest that the toxic species may not be solely limited to the free ion, but also includes ZnHPO(4)(0)(aq). Cellular uptake of ZnHPO(4)(0)(aq) through the inorganic phosphate transporter (Pit family), which requires a neutral metal phosphate complex for phosphate transport, may explain the observed toxicity. Based on visual MINTEQ (v3.0) modeling, at 50 μM total zinc, ZnHPO(4)(0)(aq) constitutes 33, 70, and 76% of the neutral metal phosphate pool at pH 6, 7, and 8, respectively. At 50 μM total zinc, cultures supplied with organic phosphate (glycerol-3-phosphate) show no significant response to pH (p = 0.13) while inhibition of inorganic phosphate-supplemented cultures, whose neutral metal phosphates are increasingly dominated by ZnHPO(4)(0)(aq), show significant pH dependence (p = 9.45 × 10(-7)). Using sodium to decrease the distribution of ZnHPO(4)(0)(aq) in the neutral metal phosphate pool also decreased the pH dependent toxicity, further supporting this mechanism. These findings show the important role of minor zinc species in organism toxicity and have wider implications because the Pit inorganic phosphate transport system is widely distributed in Bacteria, Archaea, and Eukarya.

Microbial Community Succession during Lactate Amendment and Electron Acceptor Limitation Reveals a Predominance of Metal-Reducing Pelosinus spp.

ABSTRACT The determination of the success of in situ bioremediation strategies is complex. By using controlled laboratory conditions, the influence of individual variables, such as U(VI), Cr(VI), and electron donors and acceptors on community structure, dynamics, and the metal-reducing potential can be studied. Triplicate anaerobic, continuous-flow reactors were inoculated with Cr(VI)-contaminated groundwater from the Hanford, WA, 100-H area, amended with lactate, and incubated for 95 days to obtain stable, enriched communities. The reactors were kept anaerobic with N2 gas (9 ml/min) flushing the headspace and were fed a defined medium amended with 30 mM lactate and 0.05 mM sulfate with a 48-h generation time. The resultant diversity decreased from 63 genera within 12 phyla to 11 bacterial genera (from 3 phyla) and 2 archaeal genera (from 1 phylum). Final communities were dominated by Pelosinus spp. and to a lesser degree, Acetobacterium spp., with low levels of other organisms, including methanogens. Four new strains of Pelosinus were isolated, with 3 strains being capable of Cr(VI) reduction while one also reduced U(VI). Under limited sulfate, it appeared that the sulfate reducers, including Desulfovibrio spp., were outcompeted. These results suggest that during times of electron acceptor limitation in situ, organisms such as Pelosinus spp. may outcompete the more-well-studied organisms while maintaining overall metal reduction rates and extents. Finally, lab-scale simulations can test new strategies on a smaller scale while facilitating community member isolation, so that a deeper understanding of community metabolism can be revealed.

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.

Role of Morphological Growth State and Gene Expression in Desulfovibrio africanus Strain Walvis Bay Mercury Methylation

The biogeochemical transformations of mercury are a complex process, with the production of methylmercury, a potent human neurotoxin, repeatedly demonstrated in sulfate- and Fe(III)-reducing as well as methanogenic bacteria. However, little is known regarding the morphology, genes, or proteins involved in methylmercury generation. Desulfovibrio africanus strain Walvis Bay is a Hg-methylating δ-proteobacterium with a sequenced genome and has unusual pleomorphic forms. In this study, a relationship between the pleomorphism and Hg methylation was investigated. Proportional increases in the sigmoidal (regular) cell form corresponded with increased net MeHg production but decreased when the pinched cocci (persister) form became the major morphotype. D. africanus microarrays indicated that the ferrous iron transport genes (feoAB), as well as ribosomal genes and several genes whose products are predicted to have metal binding domains (CxxC), were up-regulated during exposure to Hg in the exponential phase. Whereas no specific methylation pathways were identified, the finding that Hg may interfere with iron transport and the correlation of growth-phase-dependent morphology with MeHg production are notable. The identification of these relationships between differential gene expression, morphology, and the growth-phase dependence of Hg transformations suggests that actively growing cells are primarily responsible for methylation, and so areas with ample carbon and electron-acceptor concentrations may also generate a higher proportion of methylmercury than more oligotrophic environments. The observation of increased iron transporter expression also suggests that Hg methylation may interfere with iron biogeochemical cycles.

The toxicity of lead to Desulfovibrio desulfuricans G20 in the presence of goethite and quartz

An aqueous mixture of goethite, quartz, and lead chloride (PbCl2) was treated with the sulfate‐reducing bacterium, Desulfovibrio desulfuricans G20 (D. desulfuricans G20), in a medium specifically designed to assess metal toxicity. In the presence of 26 μM of soluble Pb, together with the goethite and quartz, D. desulfuricans G20 grew after a lag time of 5 days compared to 2 days in Pb‐, goethite‐, and quartz‐free treatments. In the absence of goethite and quartz, however, with 26 μM soluble Pb, no measurable growth was observed. Results showed that D. desulfuricans G20 first removed Pb from solutions then growth began resulting in black precipitates of Pb and iron sulfides. Transmission electron microscopic analyses of thin sections of D. desulfuricans G20 treated with 10 μM PbCl2 in goethite‐ and quartz‐free treatment showed the presence of a dense deposit of lead sulfide precipitates both in the periplasm and cytoplasm. However, thin sections of D. desulfuricans G20 treated with goethite, quartz, and PbCl2 (26 μM soluble Pb) showed the presence of a dense deposit of iron sulfide precipitates both in the periplasm and cytoplasm. Energy‐dispersive X‐ray spectroscopy, selected area electron diffraction patterns, or X‐ray diffraction analyses confirmed the structure of precipitated Pb inside the cell as galena (PbS) in goethite‐ and quartz‐free treatments, and iron sulfides in treatments with goethite, quartz, and PbCl2. Overall results suggest that even at the same soluble Pb concentration (26 μM), in the presence of goethite and quartz, apparent Pb toxicity to D. desulfuricans G20 decreased significantly. Further, accumulation of lead/iron sulfides inside D. desulfuricans G20 cells depended on the presence of goethite and quartz. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Carbon Amendments Alter Microbial Community Structure and Net Mercury Methylation Potential in Sediments

ABSTRACT Neurotoxic methylmercury (MeHg) is produced by anaerobic Bacteria and Archaea possessing the genes hgcAB, but it is unknown how organic substrate and electron acceptor availability impacts the distribution and abundance of these organisms. We evaluated the impact of organic substrate amendments on mercury (Hg) methylation rates, microbial community structure, and the distribution of hgcAB+ microbes with sediments. Sediment slurries were amended with short-chain fatty acids, alcohols, or a polysaccharide. Minimal increases in MeHg were observed following lactate, ethanol, and methanol amendments, while a significant decrease (∼70%) was observed with cellobiose incubations. Postincubation, microbial diversity was assessed via 16S rRNA amplicon sequencing. The presence of hgcAB+ organisms was assessed with a broad-range degenerate PCR primer set for both genes, while the presence of microbes in each of the three dominant clades of methylators (Deltaproteobacteria, Firmicutes, and methanogenic Archaea) was measured with clade-specific degenerate hgcA quantitative PCR (qPCR) primer sets. The predominant microorganisms in unamended sediments consisted of Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. Clade-specific qPCR identified hgcA+ Deltaproteobacteria and Archaea in all sites but failed to detect hgcA+ Firmicutes. Cellobiose shifted the communities in all samples to ∼90% non-hgcAB-containing Firmicutes (mainly Bacillus spp. and Clostridium spp.). These results suggest that either expression of hgcAB is downregulated or, more likely given the lack of 16S rRNA gene presence after cellobiose incubation, Hg-methylating organisms are largely outcompeted by cellobiose degraders or degradation products of cellobiose. These results represent a step toward understanding and exploring simple methodologies for controlling MeHg production in the environment. IMPORTANCE Methylmercury (MeHg) is a neurotoxin produced by microorganisms that bioacummulates in the food web and poses a serious health risk to humans. Currently, the impact that organic substrate or electron acceptor availability has on the mercury (Hg)-methylating microorganisms is unclear. To study this, we set up microcosm experiments exposed to different organic substrates and electron acceptors and assayed for Hg methylation rates, for microbial community structure, and for distribution of Hg methylators. The sediment and groundwater was collected from East Fork Poplar Creek in Oak Ridge, TN. Amendment with cellobiose (a lignocellulosic degradation by-product) led to a drastic decrease in the Hg methylation rate compared to that in an unamended control, with an associated shift in the microbial community to mostly nonmethylating Firmicutes. This, along with previous Hg-methylating microorganism identification methods, will be important for identifying strategies to control MeHg production and inform future remediation strategies.