Simona Rossetti

Magnetite Particles Triggering a Faster and More Robust Syntrophic Pathway of Methanogenic Propionate Degradation

Interspecies electron transfer mechanisms between Bacteria and Archaea play a pivotal role during methanogenic degradation of organic matter in natural and engineered anaerobic ecosystems. Growing evidence suggests that in syntrophic communities electron transfer does not rely exclusively on the exchange of diffusible molecules and energy carriers such as hydrogen or formate, rather microorganisms have the capability to exchange metabolic electrons in a more direct manner. Here, we show that supplementation of micrometer-size magnetite (Fe3O4) particles to a methanogenic sludge enhanced (up to 33%) the methane production rate from propionate, a key intermediate in the anaerobic digestion of organic matter and a model substrate to study energy-limited syntrophic communities. The stimulatory effect most probably resulted from the establishment of a direct interspecies electron transfer (DIET), based on magnetite particles serving as electron conduits between propionate-oxidizing acetogens and carbon dioxide-reducing methanogens. Theoretical calculations revealed that DIET allows electrons to be transferred among syntrophic partners at rates which are substantially higher than those attainable via interspecies H2 transfer. Besides the remarkable potential for improving anaerobic digestion, which is a proven biological strategy for renewable energy production, the herein described conduction-based DIET could also have a role in natural methane emissions from magnetite-rich soils and sediments.

Microbial reductive dechlorination of trichloroethene to ethene with electrodes serving as electron donors without the external addition of redox mediators.

In situ bioremediation of industrial chlorinated solvents, such as trichloroethene (TCE), is typically accomplished by providing an organic electron donor to naturally occurring dechlorinating populations. In the present study, we show that TCE dechlorinating bacteria can access the electrons required for TCE dechlorination directly from a negatively polarized (−450 mV vs. SHE) carbon paper electrode. In replicated batch experiments, a mixed dechlorinating culture, also containing Dehalococcoides spp., dechlorinated TCE to cis‐dichloroethene (cis‐DCE) and lower amounts of vinyl chloride (VC) and ethene using the polarized electrode as the sole electron donor. Conversely, neither VC nor ethene formation occurred when a pure culture of the electro‐active microorganism Geobacter lovleyi was used, under identical experimental conditions. Cyclic voltammetry tests, carried out on the filter‐sterilized supernatant of the mixed culture revealed the presence of a self‐produced redox mediator, exhibiting a midpoint potential of around −400 mV (vs. SHE). This yet unidentified redox‐active molecule appeared to be involved in the extracellular electron transfer from the electrode to the dechlorinating bacteria. The ability of dechlorinating bacteria to use electrodes as electron donors opens new perspectives for the development of clean, versatile, and efficient bioremediation systems based on a controlled subsurface delivery of electrons in support of biodegradative metabolisms and provides further evidence on the possibility of using conductive materials to manipulate and control a range of microbial bioprocesses. Biotechnol. Bioeng. 2009;103: 85–91.

Characterization of an electro-active biocathode capable of dechlorinating trichloroethene and cis-dichloroethene to ethene

In the presence of suitable electron donors, the industrial solvent trichloroethene (TCE) is reductively dechlorinated by anaerobic microorganisms, eventually to harmless ethene. In this study we investigated the use of a carbon paper electrode, polarized to -550 mV vs. standard hydrogen electrode (SHE), as direct electron donor for the mediator-less microbial reductive dechlorination of TCE to ethene. In potentiostatic batch assays, TCE was dechlorinated to predominantly cis-dichloroethene (cis-DCE) and lower amounts of vinyl chloride (VC) and ethene, at rates falling in the range 14.2-22.4 micro equiv./Ld. When cis-DCE was spiked to the system, it was also dechlorinated, to VC and ethene, but at a much lower rate (1.5-1.7 micro equiv./Ld). Scanning electron microscopy and FISH analyses revealed that the electrode was homogeneously colonized by active bacterial cells, each in direct contact with the electrode surface. Cyclic voltammetry tests revealed the presence, at the electrode interface, of formed redox active components possibly involved in the extracellular electron transfer processes, that were however detached by a vigorous magnetic stirring. Electrochemical impedance spectroscopy (EIS) tests revealed that polarization resistances of the electrode in the presence of microorganisms (ranging from 0.09 to 0.17 k Omega/cm(2)) were one-order of magnitude lower than those measured with abiotic electrodes (ranging from 1.4 to 1.8 k Omega/cm(2)). This confirmed that attached dechlorinating microorganisms significantly enhanced the kinetics of the electron transfer reactions. Thus, for the first time, the bio-electrochemical dechlorination of TCE to ethene is obtained without the apparent requirements for exogenous or self-produced redox mediators. Accordingly, this work further expands the range of metabolic reactions and microorganisms that can be stimulated by using solid-state electrodes, and has practical implications for the in situ bioremediation of groundwater contaminated by chlorinated solvents.

Phylogenetic Characterization and In Situ Detection of a Cytophaga-Flexibacter-Bacteroides Phylogroup Bacterium in Tuber borchii Vittad. Ectomycorrhizal Mycelium

ABSTRACT Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5′ and 3′ ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to theCytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genusTuber.

Bioelectrochemical hydrogen production with hydrogenophilic dechlorinating bacteria as electrocatalytic agents

Hydrogenophilic dechlorinating bacteria were shown to catalyze H(2) production by proton reduction, with electrodes serving as electron donors, either in the presence or in the absence of a redox mediator. In the presence of methyl viologen, Desulfitobacterium- and Dehalococcoides-enriched cultures produced H(2) at rates as high as 12.4 μeq/mgVSS (volatile suspended solids)/d, with the cathode set at -450 mV vs. the standard hydrogen electrode (SHE), hence very close to the reversible H(+)/H(2) potential value of -414 mV at pH 7. Notably, the Desulfitobacterium-enriched culture was capable of catalyzing H(2) production without mediators at cathode potentials lower than -700 mV. At -750 mV, the H(2) production rate with Desulfitobacterium spp. was 13.5 μeq/mgVSS/d (or 16 μeq/cm(2)/d), nearly four times higher than that of the abiotic controls. Overall, this study suggests the possibility of employing dechlorinating bacteria as hydrogen catalysts in new energy technologies such as microbial electrolysis cells.

In situ groundwater and sediment bioremediation: barriers and perspectives at European contaminated sites

This paper contains a critical examination of the current application of environmental biotechnologies in the field of bioremediation of contaminated groundwater and sediments. Based on analysis of conventional technologies applied in several European Countries and in the US, scientific, technical and administrative barriers and constraints which still need to be overcome for an improved exploitation of bioremediation are discussed. From this general survey, it is evident that in situ bioremediation is a highly promising and cost-effective technology for remediation of contaminated soil, groundwater and sediments. The wide metabolic diversity of microorganisms makes it applicable to an ever-increasing number of contaminants and contamination scenarios. On the other hand, in situ bioremediation is highly knowledge-intensive and its application requires a thorough understanding of the geochemistry, hydrogeology, microbiology and ecology of contaminated soils, groundwater and sediments, under both natural and engineered conditions. Hence, its potential still remains partially unexploited, largely because of a lack of general consensus and public concerns regarding the lack of effectiveness and control, poor reliability, and possible occurrence of side effects, for example accumulation of toxic metabolites and pathogens. Basic, applied and pre-normative research are all needed to overcome these barriers and make in situ bioremediation more reliable, robust and acceptable to the public, as well as economically more competitive. Research efforts should not be restricted to a deeper understanding of relevant microbial reactions, but also include their interactions with the large array of other relevant phenomena, as a function of the truly variable site-specific conditions. There is a need for a further development and application of advanced biomolecular tools for site investigation, as well as of advanced metabolic and kinetic modelling tools. These would allow a quicker evaluation of the bioremediation potential of a site, and in turn a preliminary assessment of the technical feasibility of the chosen bioprocess which could replace or at least reduce the need for time-consuming and expensive field tests. At the same time, field tests will probably remain unavoidable for a detailed design of full scale remedial actions and the above reported tools will in any event be useful for a better design and a more reliable operation.

Kinetics of denitrification reactions in single sludge systems

Abstract This paper reports denitrification studies performed using the anoxic reactor of a laboratory scale anoxic-aerobic plant as a batch reactor of variable volume. This was achieved by adding to the anoxic reactor a supplementary flow of nitrate after the shut down of the recirculation line and the interruption of the hydraulic connection to the aerobic reactor. By operating in this way, in a relatively short time, it is possible to get a number of experimental data sufficient to describe the biological process kinetics. The system is extremely flexible and gives kinetic data in short times for different experimental conditions. In fact, it is possible to operate at different COD/NO 3 -N ratios simply by changing the influent wastewater flowrate to the anoxic reactor. Two series of tests were performed: in the first series (use of endogenous carbon) a supplementary flow of nitrate was fed to the anoxic reactor while the wastewater influent flow was interrupted; in the second series (use of internal carbon) the influent wastewater flow was fed during the addition of nitrate. The importance of the carbonaceous substrate nature on the denitrification rate was also verified. Data analysis was performed by utilizing the integral method procedure and a zero order kinetics referring to both the substrates COD and nitrate nitrogen was considered. A satisfactory agreement between predicted and experimental data was found. Values obtained for k D range from 0.07 mg NO 3 -N/mg VSS·d, at which the carbon source is mostly endogenous, to 0.25 mg NO 3 -N/mg VSS·d, at which the carbon source consists mainly of readily biodegradable COD. Intermediate values occur when the readily biodegradable COD is limiting and denitrification takes place by utilizing the slowly biodegradable one.

Anaerobic arsenite oxidation with an electrode serving as the sole electron acceptor: A novel approach to the bioremediation of arsenic-polluted groundwater

Arsenic contamination of soil and groundwater is a serious problem worldwide. Here we show that anaerobic oxidation of As(III) to As(V), a form which is more extensively and stably adsorbed onto metal-oxides, can be achieved by using a polarized (+497 mV vs. SHE) graphite anode serving as terminal electron acceptor in the microbial metabolism. The characterization of the microbial populations at the electrode, by using in situ detection methods, revealed the predominance of gammaproteobacteria. In principle, the proposed bioelectrochemical oxidation process would make it possible to provide As(III)-oxidizing microorganisms with a virtually unlimited, low-cost and low-maintenance electron acceptor as well as with a physical support for microbial attachment.

High frequency ultrasound pretreatment for sludge anaerobic digestion: Effect on floc structure and microbial population

In this work the potential of high frequency ultrasounds as pretreatment for sludge anaerobic digestion has been assessed. Irradiation with 200kHz ultrasounds was efficient in disintegrating the floc structure increasing the available fraction of soluble organic matter (up to seven times at 25,000kJ/kgTS). Batch anaerobic digestion tests were carried out on lab-scale reactors fed either with untreated or disintegrated sludge inoculated with anaerobic sludge, at different feed/inoculum ratio (F/I=0.5 and 1). Degradation of particulate matter, biogas production and related microbial community composition (estimated by fluorescence in situ hybridization, FISH) were investigated. Sludge ultrasounds pretreatment led to an overall improvement of the digestion performances, with a maximum biogas gain of 40% at F/I=0.5. FISH showed a key-role of Methanosarcina spp. in the main reactions of biogas synthesis.

Structure analysis and performance of a microbial community from a contaminated aquifer involved in the complete reductive dechlorination of 1,1,2,2-tetrachloroethane to ethene.

An anaerobic microcosm set up with aquifer material from a 1,1,2,2‐tetrachloroethane (TeCA) contaminated site and amended with butyrate showed a complete TeCA dechlorination to ethene. A structure analysis of the microbial community was performed by fluorescence in situ hybridization (FISH) with already available and on purpose designed probes from sequences retrieved through 16S rDNA clone library construction. FISH was chosen as identification tool to evaluate in situ whether the retrieved sequences belong to primary bacteria responsible for the biodegradative reactions. FISH probes identified up to 80% of total bacteria and revealed the absence or the marginal presence of known TeCA degraders and the abundance of two well‐known H2‐utilizing halorespiring bacteria, Sulfurospirillum (32.4 ± 8.6% of total bacteria) and Dehalococcoides spp. (14.8 ± 2.8), thereby providing a strong indication of their involvement in the dechlorination processes. These results were supported by the kinetic and thermodynamic analysis which provided indications that hydrogen was the actual electron donor for TeCA dechlorination. The specific probes, developed in this study, for known dechlorinators (i.e., Geobacter, Dehalobacter, and Sulfurospirillum species) represent a valuable tool for any future in situ bioremediation study as well as a quick and specific investigation tool for tracking their distribution in the field. Biotechnol. Bioeng. 2008;99: 240–249.