Flynn W. Picardal

Methane-Producing Microbial Community in a Coal Bed of the Illinois Basin

ABSTRACT A series of molecular and geochemical studies were performed to study microbial, coal bed methane formation in the eastern Illinois Basin. Results suggest that organic matter is biodegraded to simple molecules, such as H2 and CO2, which fuel methanogenesis and the generation of large coal bed methane reserves. Small-subunit rRNA analysis of both the in situ microbial community and highly purified, methanogenic enrichments indicated that Methanocorpusculum is the dominant genus. Additionally, we characterized this methanogenic microorganism using scanning electron microscopy and distribution of intact polar cell membrane lipids. Phylogenetic studies of coal water samples helped us develop a model of methanogenic biodegradation of macromolecular coal and coal-derived oil by a complex microbial community. Based on enrichments, phylogenetic analyses, and calculated free energies at in situ subsurface conditions for relevant metabolisms (H2-utilizing methanogenesis, acetoclastic methanogenesis, and homoacetogenesis), H2-utilizing methanogenesis appears to be the dominant terminal process of biodegradation of coal organic matter at this location.

Repeated anaerobic microbial redox cycling of iron.

ABSTRACT Some nitrate- and Fe(III)-reducing microorganisms are capable of oxidizing Fe(II) with nitrate as the electron acceptor. This enzymatic pathway may facilitate the development of anaerobic microbial communities that take advantage of the energy available during Fe-N redox oscillations. We examined this phenomenon in synthetic Fe(III) oxide (nanocrystalline goethite) suspensions inoculated with microflora from freshwater river floodplain sediments. Nitrate and acetate were added at alternate intervals in order to induce repeated cycles of microbial Fe(III) reduction and nitrate-dependent Fe(II) oxidation. Addition of nitrate to reduced, acetate-depleted suspensions resulted in rapid Fe(II) oxidation and accumulation of ammonium. High-resolution transmission electron microscopic analysis of material from Fe redox cycling reactors showed amorphous coatings on the goethite nanocrystals that were not observed in reactors operated under strictly nitrate- or Fe(III)-reducing conditions. Microbial communities associated with N and Fe redox metabolism were assessed using a combination of most-probable-number enumerations and 16S rRNA gene analysis. The nitrate-reducing and Fe(III)-reducing cultures were dominated by denitrifying Betaproteobacteria (e.g., Dechloromonas) and Fe(III)-reducing Deltaproteobacteria (Geobacter), respectively; these same taxa were dominant in the Fe cycling cultures. The combined chemical and microbiological data suggest that both Geobacter and various Betaproteobacteria participated in nitrate-dependent Fe(II) oxidation in the cycling cultures. Microbially driven Fe-N redox cycling may have important consequences for both the fate of N and the abundance and reactivity of Fe(III) oxides in sediments.

Chemical and Biological Interactions during Nitrate and Goethite Reduction by Shewanella putrefaciens 200

ABSTRACT Although previous research has demonstrated that NO3− inhibits microbial Fe(III) reduction in laboratory cultures and natural sediments, the mechanisms of this inhibition have not been fully studied in an environmentally relevant medium that utilizes solid-phase, iron oxide minerals as a Fe(III) source. To study the dynamics of Fe and NO3− biogeochemistry when ferric (hydr)oxides are used as the Fe(III) source, Shewanella putrefaciens 200 was incubated under anoxic conditions in a low-ionic-strength, artificial groundwater medium with various amounts of NO3− and synthetic, high-surface-area goethite. Results showed that the presence of NO3− inhibited microbial goethite reduction more severely than it inhibited microbial reduction of the aqueous or microcrystalline sources of Fe(III) used in other studies. More interestingly, the presence of goethite also resulted in a twofold decrease in the rate of NO3− reduction, a 10-fold decrease in the rate of NO2− reduction, and a 20-fold increase in the amounts of N2O produced. Nitrogen stable isotope experiments that utilized δ15N values of N2O to distinguish between chemical and biological reduction of NO2− revealed that the N2O produced during NO2− or NO3− reduction in the presence of goethite was primarily of abiotic origin. These results indicate that concomitant microbial Fe(III) and NO3− reduction produces NO2− and Fe(II), which then abiotically react to reduce NO2− to N2O with the subsequent oxidation of Fe(II) to Fe(III).

Abiotic and Microbial Interactions during Anaerobic Transformations of Fe(II) and NOX

Microbial Fe(II) oxidation using NO3- as the terminal electron acceptor [nitrate-dependent Fe(II) oxidation, NDFO] has been studied for over 15 years. Although there are reports of autotrophic isolates and stable enrichments, many of the bacteria capable of NDFO are known organotrophic NO3--reducers that require the presence of an organic, primary substrate, e.g., acetate, for significant amounts of Fe(II) oxidation. Although the thermodynamics of Fe(II) oxidation are favorable when coupled to either NO3- or NO2- reduction, the kinetics of abiotic Fe(II) oxidation by NO3- are relatively slow except under special conditions. NDFO is typically studied in batch cultures containing millimolar concentrations of Fe(II), NO3-, and the primary substrate. In such systems, NO2- is often observed to accumulate in culture media during Fe(II) oxidation. Compared to NO3-, abiotic reactions of biogenic NO2- and Fe(II) are relatively rapid. The kinetics and reaction pathways of Fe(II) oxidation by NO2- are strongly affected by medium composition and pH, reactant concentration, and the presence of Fe(II)-sorptive surfaces, e.g., Fe(III) oxyhydroxides and cellular surfaces. In batch cultures, the combination of abiotic and microbial Fe(II) oxidation can alter product distribution and, more importantly, results in the formation of intracellular precipitates and extracellular Fe(III) oxyhydroxide encrustations that apparently limit further cell growth and Fe(II) oxidation. Unless steps are taken to minimize or account for potential abiotic reactions, results of microbial NDFO studies can be obfuscated by artifacts of the chosen experimental conditions, the use of inappropriate analytical methods, and the resulting uncertainties about the relative importance of abiotic and microbial reactions. In this manuscript, abiotic reactions of NO3- and NO2- with aqueous Fe2+, chelated Fe(II), and solid-phase Fe(II) are reviewed along with factors that can influence overall NDFO reaction rates in microbial systems. In addition, the use of low substrate concentrations, continuous-flow systems, and experimental protocols that minimize experimental artifacts and reduce the potential for under- or overestimation of microbial NDFO rates are discussed.

Enhanced Growth of Acidovorax sp. Strain 2AN during Nitrate-Dependent Fe(II) Oxidation in Batch and Continuous-Flow Systems

ABSTRACT Microbial nitrate-dependent, Fe(II) oxidation (NDFO) is a ubiquitous biogeochemical process in anoxic sediments. Since most microorganisms that can oxidize Fe(II) with nitrate require an additional organic substrate for growth or sustained Fe(II) oxidation, the energetic benefits of NDFO are unclear. The process may also be self-limiting in batch cultures due to formation of Fe-oxide cell encrustations. We hypothesized that NDFO provides energetic benefits via a mixotrophic physiology in environments where cells encounter very low substrate concentrations, thereby minimizing cell encrustations. Acidovorax sp. strain 2AN was incubated in anoxic batch reactors in a defined medium containing 5 to 6 mM NO3 −, 8 to 9 mM Fe2+, and 1.5 mM acetate. Almost 90% of the Fe(II) was oxidized within 7 days with concomitant reduction of nitrate and complete consumption of acetate. Batch-grown cells became heavily encrusted with Fe(III) oxyhydroxides, lost motility, and formed aggregates. Encrusted cells could neither oxidize more Fe(II) nor utilize further acetate additions. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced, and further additions of acetate and Fe(II)-EDTA could be oxidized. Experiments using a novel, continuous-flow culture system with low concentrations of substrate, e.g., 100 μM NO3 −, 20 μM acetate, and 50 to 250 μM Fe2+, showed that the growth yield of Acidovorax sp. strain 2AN was always greater in the presence of Fe(II) than in its absence, and electron microscopy showed that encrustation was minimized. Our results provide evidence that, under environmentally relevant concentrations of substrates, NDFO can enhance growth without the formation of growth-limiting cell encrustations.

Inhibition of NO3- and NO2- reduction by microbial Fe(III) reduction: evidence of a reaction between NO2- and cell surface-bound Fe2+.

ABSTRACT A recent study (D. C. Cooper, F. W. Picardal, A. Schimmelmann, and A. J. Coby, Appl. Environ. Microbiol. 69:3517-3525, 2003) has shown that NO3− and NO2− (NOx−) reduction by Shewanella putrefaciens 200 is inhibited in the presence of goethite. The hypothetical mechanism offered to explain this finding involved the formation of a Fe(III) (hydr)oxide coating on the cell via the surface-catalyzed, abiotic reaction between Fe2+ and NO2−. This coating could then inhibit reduction of NOx− by physically blocking transport into the cell. Although the data in the previous study were consistent with such an explanation, the hypothesis was largely speculative. In the current work, this hypothesis was tested and its environmental significance explored through a number of experiments. The inhibition of ∼3 mM NO3− reduction was observed during reduction of a variety of Fe(III) (hydr)oxides, including goethite, hematite, and an iron-bearing, natural sediment. Inhibition of oxygen and fumarate reduction was observed following treatment of cells with Fe2+ and NO2−, demonstrating that utilization of other soluble electron acceptors could also be inhibited. Previous adsorption of Fe2+ onto Paracoccus denitrificans inhibited NOx− reduction, showing that Fe(II) can reduce rates of soluble electron acceptor utilization by non-iron-reducing bacteria. NO2− was chemically reduced to N2O by goethite or cell-sorbed Fe2+, but not at appreciable rates by aqueous Fe2+. Transmission and scanning electron microscopy showed an electron-dense, Fe-enriched coating on cells treated with Fe2+ and NO2−. The formation and effects of such coatings underscore the complexity of the biogeochemical reactions that occur in the subsurface.

Microbial growth on dichlorobiphenyls chlorinated on both rings as a sole carbon and energy source.

ABSTRACT We have isolated bacterial strains capable of aerobic growth onortho-substituted dichlorobiphenyls as sole carbon and energy sources. During growth on 2,2′-dichlorobiphenyl and 2,4′-dichlorobiphenyl strain SK-4 produced stoichiometric amounts of 2-chlorobenzoate and 4-chlorobenzoate, respectively. Chlorobenzoates were not produced when strain SK-3 was grown on 2,4′-dichlorobiphenyl.

Induction of Nitrate-Dependent Fe(II) Oxidation by Fe(II) in Dechloromonas sp. Strain UWNR4 and Acidovorax sp. Strain 2AN

ABSTRACT We evaluated the inducibility of nitrate-dependent Fe(II)-EDTA oxidation (NDFO) in non-growth, chloramphenicol-amended, resting-cell suspensions of Dechloromonas sp. strain UWNR4 and Acidovorax sp. strain 2AN. Cells previously incubated with Fe(II)-EDTA oxidized ca. 6-fold more Fe(II)-EDTA than cells previously incubated with Fe(III)-EDTA. This is the first report of induction of NDFO by Fe(II).

Enhanced anaerobic biotransformation of carbon tetrachloride in the presence of reduced iron oxides

Rates of anaerobic transformation of carbon tetrachloride (CT) by the facultative anaerobe Shewanella putrefaciens 200 were increased by the presence of Fe(III)-containing minerals. In batch reactors with amorphous, Fe(III)-hydroxide and S. putrefaciens, CT transformation rates could be modeled by a first-order expression in which the pseudo-first-order rate constant was linearly proportional to the initial concentration of Fe(III)-oxide. Subsequent measurement of soluble and acid-extractable Fe(II) showed that increased CT transformation rates were proportional to microbially reduced, surface-bound Fe(II), rather than soluble Fe(II). In biomimetic experiments using 20 mM dithiothreitol (DTT) as a reductant, rates of transformation of CT by DTT were low in the absence of Fe(III)-oxides. However, in the presence of iron oxides, DTT was able to transform CT at elevated rates. Results again strongly suggested that surface-bound Fe(II) was primarily responsible for the reductive transformation of CT. Results suggested that the surface area of the iron mineral determines the rate of CT transformation by affecting the extent of iron reduction. Chloroform (CF) was the only transformation product identified and production of CF was nonstoichiometric. In microbial and abiotic experiments with Fe(III) oxides, the percentage of the transformed CT recovered as CF decreased even though the rate and extent of CT transformation was increased. Overall, our results have important implications for an improved understanding of possible microbial and geochemical interactions in the environmental transformation of chlorinated organic pollutants and for modeling of CT transformation rates in anaerobic, iron-bearing sediments.

Carbon metabolism of the moderately acid-tolerant acetogen Clostridium drakei isolated from peat.

A moderately acid-tolerant, malodorous bacterium, strain FP, was isolated from peat that had a pore water pH of c. 4.2. The 16S rRNA gene sequence of FP was closely related to that of acetogens Clostridium drakei, Clostridium scatologenes, and Clostridium carboxidivorans. The DNA-DNA reassociation values obtained with DNA from FP and that of these three acetogens approximated 80%, 64%, and 59%, respectively, indicating that FP was a new strain of C. drakei. FP had broad pH and temperature ranges (3.6-7.4 and 5-40 degrees C, respectively), and metabolized a wide range of substrates, including cellobiose, glucose, xylose, vanillate, ferulate, lactate, propanol, formate, H(2)-CO(2), and CO-CO(2). Acetate was the primary reduced end product, and substrate/product stoichiometries were indicative of acetogenesis at circumneutral pH. Butyrate and H(2) became significant products from glucose at low pH. FP tolerated and could consume moderate amounts of O(2). These results (1) demonstrate that peat can harbor acetogens with a broad substrate range and tolerance to transient exposure to O(2), and (2) confirm that C. drakei, the type strain of which was originally isolated from an acidic coal mine pond, occurs in moderately acidic habitats.