Jo Philips

Carbon cloth stimulates direct interspecies electron transfer in syntrophic co-cultures.

This study investigated the possibility that the electrical conductivity of carbon cloth accelerates direct interspecies electron transfer (DIET) in co-cultures. Carbon cloth accelerated metabolism of DIET co-cultures (Geobacter metallireducens-Geobacter sulfurreducens and G.metallireducens-Methanosarcina barkeri) but did not promote metabolism of co-cultures performing interspecies H2 transfer (Desulfovibrio vulgaris-G.sulfurreducens). On the other hand, DIET co-cultures were not stimulated by poorly conductive cotton cloth. Mutant strains lacking electrically conductive pili, or pili-associated cytochromes participated in DIET only in the presence of carbon cloth. In co-cultures promoted by carbon cloth, cells were primarily associated with the cloth although the syntrophic partners were too far apart for cell-to-cell biological electrical connections to be feasible. Carbon cloth seemingly mediated interspecies electron transfer between the distant syntrophic partners. These results suggest that the ability of carbon cloth to accelerate DIET should be considered in anaerobic digester designs that incorporate carbon cloth.

A three-layer diffusion-cell to examine bio-enhanced dissolution of chloroethene dense non-aqueous phase liquid

Microbial reductive dechlorination of trichloroethene (TCE) and perchloroethene (PCE) in the vicinity of their dense non-aqueous phase liquid (DNAPL) has been shown to accelerate DNAPL dissolution. A three-layer diffusion-cell was developed to quantify this bio-enhanced dissolution and to measure the conditions near the DNAPL interface. The 12 cm long diffusion-cell setup consists of a 5.5 cm central porous layer (sand), a lower 3.5 cm DNAPL layer and a top 3 cm water layer. The water layer is frequently refreshed to remove chloroethenes at the upper boundary of the porous layer, while the DNAPL layer maintains the saturated chloroethene concentration at the lower boundary. Two abiotic and two biotic diffusion-cells with TCE DNAPL were tested. In the abiotic diffusion-cells, a linear steady state TCE concentration profile between the DNAPL and the water layer developed beyond 21 d. In the biotic diffusion-cells, TCE was completely converted into cis-dichloroethene (cis-DCE) at 2.5 cm distance of the DNAPL. Dechlorination was likely inhibited up to a distance of 1.5 cm from the DNAPL, as in this part the TCE concentration exceeded the cultures maximum tolerable concentration (2.5mM). The DNAPL dissolution fluxes were calculated from the TCE concentration gradient, measured at the interface of the DNAPL layer and the porous layer. Biotic fluxes were a factor 2.4 (standard deviation 0.2) larger than abiotic dissolution fluxes. This diffusion-cell setup can be used to study the factors affecting the bio-enhanced dissolution of DNAPL and to assess bioaugmentation, pH buffer addition and donor delivery strategies for source zones.

Inhibition of microbial trichloroethylene dechorination by Fe (III) reduction depends on Fe mineralogy: a batch study using the bioaugmentation culture KB-1

Microbial reductive dechlorination of trichloroethylene (TCE) in groundwater can be stimulated by adding of electron donors. However, side reactions such as Fe (III) reduction competes with this reaction. This study was set-up to relate the inhibition of microbial TCE dechlorination to the quantity and quality (mineralogy) of Fe (III) in the substrate and to calibrate a substrate extraction procedure for testing bioavailable Fe (III) in sediments. Batch experiments were set-up with identical inoculum (KB-1 culture) and liquid medium composition, and adding either 1) variable amounts of ferrihydrite or 2) 14 different Fe (III) minerals coated onto or mixed in with quartz sand (at constant total Fe) at a stoichiometric excess Fe (III) over electron donor. Increasing amounts of ferrihydrite significantly increased the time for complete TCE degradation from 8 days (control sand) to 28 days (excess Fe). Acid extractable Fe (II) increased and magnetite formed during incubation, confirming Fe (III) reduction. At constant total Fe in the sand, TCE dechlorination time varied with Fe mineralogy between 8 days (no Fe added) to >120 days (Fe-containing bentonite). In general, poorly crystalline Fe (III) minerals inhibited TCE dechlorination whereas crystalline Fe (III) minerals such as goethite or hematite had no effect. The TCE inhibition time was positively correlated to the Fe (II) determined after 122 days and to the surface area of the Fe (III) minerals. Only a fraction of total Fe (III) is reduced, likely because of solubility constraints and/or coating of Fe (III) minerals by Fe (II) minerals. Iron extraction tests based on Fe (III) reduction using NH2OH(.)HCl predict the competitive inhibition of TCE degradation in these model systems. This study shows that Fe mineralogy rather that total Fe content determines the competitive inhibition of TCE dechlorination.

Acidification due to microbial dechlorination near a trichloroethene DNAPL is overcome with pH buffer or formate as electron donor: experimental demonstration in diffusion- cells

Acidification due to microbial dechlorination of trichloroethene (TCE) can limit the bio-enhanced dissolution of TCE dense non-aqueous phase liquid (DNAPL). This study related the dissolution enhancement of a TCE DNAPL to the pH buffer capacity of the medium and the type of electron donor used. In batch systems, dechlorination was optimal at pH7.1-7.5, but was completely inhibited below pH6.2. In addition, dechlorination in batch systems led to a smaller pH decrease at an increasing pH buffer capacity or with the use of formate instead of lactate as electron donor. Subsequently, bio-enhanced TCE DNAPL dissolution was quantified in diffusion-cells with a 5.5 cm central sand layer, separating a TCE DNAPL layer from an aqueous top layer. Three different pH buffer capacities (2.9 mM-17.9 mM MOPS) and lactate or formate as electron donor were applied. In the lactate fed diffusion-cells, the DNAPL dissolution enhancement factor increased from 1.5 to 2.2 with an increase of the pH buffer capacity. In contrast, in the formate fed diffusion-cells, the DNAPL dissolution enhancement factor (2.4±0.3) was unaffected by the pH buffer capacity. Measurement of the pore water pH confirmed that the pH decreased less with an increased pH buffer capacity or with formate instead of lactate as electron donor. These results suggest that the significant impact of acidification on bio-enhanced DNAPL dissolution can be overcome by the amendment of a pH buffer or by applying a non acidifying electron donor like formate.

Distribution of a dechlorinating community in relation to the distance from a trichloroethene dense nonaqueous phase liquid in a model aquifer.

The toxicity of trichloroethene (TCE) likely restricts microbial activity in close vicinity of a TCE dense nonaqueous phase liquid (DNAPL). This study examined the distribution of a dechlorinating community in relation to the distance from a TCE DNAPL using a diffusion-cell set-up. Subcultures of the KB-1(™) culture dechlorinating TCE to cis-dichloroethene and grown with either formate or lactate as electron donor were used to inoculate the diffusion-cells. 16S rRNA gene clone library analysis showed that both inocula consisted of dechlorinating bacteria similar to Geobacter lovleyi SZ and fermentative microorganisms related to Clostridium and Clostridiales. qPCR and RFLP analyses of pore water and sand samples showed a stratified microbial community composition in the diffusion-cells. Geobacter dominated where TCE was present, that is, in the lower 3 cm of the 5.5-cm-thick sand layer. Even at 0.5 cm distance from the DNAPL layer, Geobacter densities were two orders of magnitude higher than at inoculation, despite the expected TCE toxicity. In the upper 2.5 cm of the sand layer, where TCE was depleted, apparently fermenting populations prevailed, corresponding to Clostridium in some diffusion-cells. This analysis demonstrates that the microbial community composition in a source zone is related to the distance from the DNAPL.

Biofilm Formation by Clostridium ljungdahlii Is Induced by Sodium Chloride Stress: Experimental Evaluation and Transcriptome Analysis

The acetogen Clostridium ljungdahlii is capable of syngas fermentation and microbial electrosynthesis. Biofilm formation could benefit both these applications, but was not yet reported for C. ljungdahlii. Biofilm formation does not occur under standard growth conditions, but attachment or aggregation could be induced by different stresses. The strongest biofilm formation was observed with the addition of sodium chloride. After 3 days of incubation, the biomass volume attached to a plastic surface was 20 times higher with than without the addition of 200 mM NaCl to the medium. The addition of NaCl also resulted in biofilm formation on glass, graphite and glassy carbon, the latter two being often used electrode materials for microbial electrosynthesis. Biofilms were composed of extracellular proteins, polysaccharides, as well as DNA, while pilus-like appendages were observed with, but not without, the addition of NaCl. A transcriptome analysis comparing planktonic (no NaCl) and biofilm (NaCl addition) cells showed that C. ljungdahlii coped with the salt stress by the upregulation of the general stress response, Na+ export and osmoprotectant accumulation. A potential role for poly-N-acetylglucosamines and D-alanine in biofilm formation was found. Flagellar motility was downregulated, while putative type IV pili biosynthesis genes were not expressed. Moreover, the gene expression analysis suggested the involvement of the transcriptional regulators LexA, Spo0A and CcpA in stress response and biofilm formation. This study showed that NaCl addition might be a valuable strategy to induce biofilm formation by C. ljungdahlii, which can improve the efficacy of syngas fermentation and microbial electrosynthesis applications.

Inhibition of Geobacter dechlorinators at elevated trichloroethene concentrations is explained by a reduced activity rather than by an enhanced cell decay.

Microbial dechlorination of trichloroethene (TCE) is inhibited at elevated TCE concentrations. A batch experiment and modeling analysis were performed to examine whether this self-inhibition is related to an enhanced cell decay or a reduced dechlorination activity at increasing TCE concentrations. The batch experiment combined four different initial TCE concentrations (1.4-3.0 mM) and three different inoculation densities (4.0 × 10(5) to 4.0 × 10(7)Geobacter cells·mL(-1)). Chlorinated ethene concentrations and Geobacter 16S rRNA gene copy numbers were measured. The time required for complete conversion of TCE to cis-DCE increased with increasing initial TCE concentration and decreasing inoculation density. Both an enhanced decay and a reduced activity model fitted the experimental results well, although the reduced activity model better described the lag phase and microbial decay in some treatments. In addition, the reduced activity model succeeded in predicting the reactivation of the dechlorination reaction in treatments in which the inhibiting TCE concentration was lowered after 80 days. In contrast, the enhanced decay model predicted a Geobacter cell density that was too low to allow recovery for these treatments. Conclusively, our results suggest that TCE self-inhibition is related to a reduced dechlorination activity rather than to an enhanced cell decay at elevated TCE concentrations.

Electron donor limitations reduce microbial enhanced trichloroethene DNAPL dissolution: A flux-based analysis using diffusion-cells

Electron donor limitations likely reduce microbial enhanced trichloroethene (TCE) dense non-aqueous phase liquid (DNAPL) dissolution. This study quantitatively examined the relation between the DNAPL dissolution enhancement and the electron donor supply rate. An experiment used diffusion-cells with a 5.5 cm central sand layer, separating a DNAPL layer from an aqueous top layer. Top layers were amended with different concentrations of formate (0-16 mM). The TCE DNAPL dissolution rate increased from no enhancement compared to abiotic dissolution without formate, to a 2.4 times dissolution enhancement with 16 mM formate amended to the top layer. With 2, 4 and 8 mM formate amended the top layer, the TCE diffusion flux out of the DNAPL layer equaled the formate diffusion flux out of the top layer, which illustrates their stoichiometric interdependence under electron donor limiting conditions. In contrast, with 16 mM formate amended to the top layer, the TCE diffusion flux was lower than the formate diffusion flux, demonstrating that the dechlorination kinetics limited the DNAPL dissolution enhancement. The DNAPL dissolution flux under electron donor limiting conditions was readily predicted from the electron donor concentration in the top layer.

Electron transfer mechanisms in biofilms

Abstract Understanding the mechanisms by which microorganisms transfer electrons to and from electrodes is essential for the development of the many potential applications of bioelectrochemical systems. The knowledge on the extracellular electron transfer (EET) mechanisms of microorganisms has steeply increased during the last decade(s) due to extensive fundamental research. In general, there are two types of EET mechanisms: (1) direct EET using outer surface redox molecules and/or conductive nanowires and (2) mediated EET using electron shuttles. Direct EET is best understood for the anode-respiring Geobacter sulfurreducens, which uses outer membrane cytochromes and pili nanowires to transport electrons to an anode. The other model electrogen, Shewanella oneidensis, mainly uses flavin shuttles to mediate its EET, but also has membrane extensions that can act as conductive nanowires. Electrogens are typically heterotrophic Fe(III) reducers, but a much wider diversity exists in electrotrophic microorganisms, as a cathode can be used as electron donor by oxygen-reducing, fumarate-reducing, nitrogen compound-reducing, hydrogen-producing, methanogenic, and acetogenic microorganisms. Nevertheless, microbial electron uptake from a cathode is still much less understood than electron transfer to anodes. Some model electrotrophs, including the acidophilic aerobe Acidithiobacillus ferrooxidans, the acetogen Clostridium ljungdahlii, and the methanogen Methanococcus maripaludis, have already been identified. Several studies have suggested a direct EET mechanism for these electrotrophs, but hydrogen-mediated EET likely also plays an important role. Besides exchanging electrons with electrodes, microorganisms can also exchange electrons between each other. Interspecies electron transfer (IET) usually relies on a mediated mechanism using hydrogen and/or formate as shuttling molecules, while cocultures with Geobacter species use a direct mechanism. Most EET mechanisms transport extracellular electrons only over micrometer-scale distances, but marine “cable bacteria” have been proven to have the exceptional capacity to transport electrons transport over centimeter-long distances. This chapter provides an overview of the current knowledge on EET mechanisms of anodic and cathodic biofilms, while also the mechanisms of IET are shortly discussed.

Motile Geobacter dechlorinators migrate into a model source zone of trichloroethene dense non-aqueous phase liquid: Experimental evaluation and modeling

Microbial migration towards a trichloroethene (TCE) dense non-aqueous phase liquid (DNAPL) could facilitate the bioaugmentation of TCE DNAPL source zones. This study characterized the motility of the Geobacter dechlorinators in a TCE to cis-dichloroethene dechlorinating KB-1(™) subculture. No chemotaxis towards or away from TCE was found using an agarose in-plug bridge method. A second experiment placed an inoculated aqueous layer on top of a sterile sand layer and showed that Geobacter migrated several centimeters in the sand layer in just 7days. A random motility coefficient for Geobacter in water of 0.24±0.02cm(2)·day(-1) was fitted. A third experiment used a diffusion-cell setup with a 5.5cm central sand layer separating a DNAPL from an aqueous top layer as a model source zone to examine the effect of random motility on TCE DNAPL dissolution. With top layer inoculation, Geobacter quickly colonized the sand layer, thereby enhancing the initial TCE DNAPL dissolution flux. After 19days, the DNAPL dissolution enhancement was only 24% lower than with an homogenous inoculation of the sand layer. A diffusion-motility model was developed to describe dechlorination and migration in the diffusion-cells. This model suggested that the fast colonization of the sand layer by Geobacter was due to the combination of random motility and growth on TCE.