Impact of iron reduction on the metabolism of Clostridium acetobutylicum

Iron is essential for most living organisms. In addition, its biogeochemical cycling influences important processes in the geosphere (e.g., the mobilization or immobilization of trace elements and contaminants). The reduction of Fe(III) to Fe(II) can be catalysed microbially, particularly by metal-respiring bacteria utilizing Fe(III) as a terminal electron acceptor. Furthermore, Grampositive fermentative iron reducers are known to reduce Fe(III) by using it as a sink for excess reducing equivalents, as a form of enhanced fermentation. Here, we use the Gram-positive fermentative bacterium Clostridium acetobutylicum as a model system due to its ability to reduce heavy metals.We investigated the reduction of soluble and solid iron during fermentation. We found that exogenous (resazurin, resorufin, anthraquinone-2,- 6-disulfonate) aswell as endogenous (riboflavin) electron mediators enhance solid iron reduction. In addition, iron reduction buffers the pH, and elicits a shift in the carbon and electron flow to less reduced products relative to fermentation. This study underscores the role fermentative bacteria can play in iron cycling and provides insights into the metabolic profile of coupled fermentation and iron reduction with laboratory experiments and metabolic network modelling

Mechanism(s) of electron transfer and metabolic changes during iron reduction by Clostridium acetobutylicum

Cornelia List

Microorganisms catalyze many of the redox transformations driving the iron cycle in the geosphere. For instance, iron cycling includes the reduction of solid-phase and soluble ferric iron (Fe(III)) to ferrous iron (Fe(II)). However, in order to reduce solid-phase Fe(III), the reduction requires the delivery of electrons extracellularly, requiring a specialized electron transfer system. The mechanism of iron reduction by Gram-negative bacteria has been de-scribed in some detail, but very little is known for Gram-positive bacteria. These microorgan-isms are likely to contribute to iron reduction significantly, particularly in environments where Gram-negative bacteria fare poorly, such as metal contaminated sites, or hydrother-mal environments. Some Gram-positive bacteria use metal reduction as a form of enhanced fermentation, where the metal is a sink for excess reducing equivalents. In these cases, metal reduction is facultative and is not a mechanism for direct energy conservation. Here, we use Clostridium acetobutylicum, a Gram-positive fermenter, as a model organism for its ability to reduce iron and contaminant heavy metals. A major advantage of this model organism is that genetic tools are available to study genes involved in the metal reduction process. We investigated the reduction of solid-phase and soluble Fe(III) by C. acetobutyli-cum and found that it reduces both forms of iron. The bacteria do not require direct contact with solid-phase Fe(III) (as hydrous ferric oxide, HFO) for the reduction to occur. In fact, flavins were identified as likely to be involved in the extracellular electron transfer (EET) in this organism. We found that HFO reduction is increased by utilizing exogenous (resazurin, resorufin, anthraquinone-2,6-disulfonate) or endogenous (riboflavin, RF) electron shuttles. Inactivation of a putative flavin transporter (CAC2841) decreased HFO and Fe(III)-citrate reduction. We proposed that a complex CAC2841/ CAC3100/ CAC3101/ CAC3102 imports RF and CAC2841/ CAC3100 exports flavin adenine dinucleotide (FAD). FAD might be hy-drolyzed by CAC2761 and CAC2766, which interact with membrane-anchored flavoproteins potentially involved in EET (CAC2762 and CAC2767). Additionally, we performed a transcriptomic analysis of Fe(III)-citrate- and HFO-reducing cultures and compared them to fermentation alone. We found two Fe-S oxidoreductases (CA1254 and CAC1021) upregulated with Fe(III)-citrate, which have an unknown function and could be putative Fe(III)-citrate reductases. Additional upregulated redox active pro-teins with potential involvement in solid-phase Fe(III) reduction were identified as CA1044 and CAC3371. These proteins should be considered for further studies. Furthermore, iron reduction impacts bacterial metabolism by buffering the pH and shifting the carbon and electron flow to less reduced metabolites as compared to fermentation. In addition to catabolic pathways, metabolic modeling showed that iron reduction influences anabolic pathways. Finally, metabolic modeling suggests that NADH is the physiological elec-tron donor for iron reduction in this system. This study contributes to a better understanding of the mechanism of iron reduction by Gram-positive bacteria and provides implications for further studies to understand the EET by fermenters in natural environments.

EPFL2019

Metal reduction and sporulation in Desulfotomaculum reducens

Elena Cristina Dalla Vecchia

The genera Desulfotomaculum and Clostridium, belonging to the phylum Firmicutes, comprise Gram-positive, low G+C genomic content, anaerobic, spore- forming bacteria. Desulfotomaculum is a metabolically and environmentally versatile genus capable of growing fermentatively as well as by respiring sulfate. D. reducens is also capable of metal reduction and has been reported to conserve energy from this process. In this thesis, iron (Fe(III)) reduction by D. reducens in the presence of pyruvate or lactate as electron donors was investigated. When pyruvate is present, D. reducens grows fermentatively. If Fe(III) is present, it can act as an electron sink alternative to protons and consume excess reducing equivalents accumulated during pyruvate oxidation. Electron transfer to Fe(III) by D. reducens in the presence of pyruvate is mediated by a soluble electron carrier, likely riboflavin, released by cells during fermentation. This mechanism allows the microorganism to reduce extracellular, solid phase Fe(III) even if it is not directly accessible by physical contact. In the presence of lactate, D. reducens is unable to conserve significant energy from electron donor oxidation and Fe(III) reduction. In contrast to pyruvate, during lactate oxidation, no redox-active compound is released by the cells, and extracellular, solid phase Fe(III) can only be reduced by direct contact. This implies that a surface exposed reductase must be present in D. reducens cells. The investigation of the surface proteome of this organism led to the identification of three proteins, most likely localized to the cell surface, potentially involved in Fe(III) reduction. One is a heterodisulfide reductase subunit A, a putative intermediate in the Fe(III) reduction chain. The second is a membrane-associated hydrogenase. The third protein is annotated as alkyl hydroperoxide reductase although its actual function may be thiol-disulfide oxidoreduction; several lines of evidence suggest the involvement of the latter in metal reduction by D. reducens. Clostridium species are widespread in the environment and are capable of utilizing a great variety of organic substrates for fermentative growth, while they are unable to conserve energy through respiration. However, they can reduce terminal electron acceptors to eliminate excess reducing equivalents from fermentation. Among the electron acceptors used by Clostridium spp., are metals, such as iron and uranium (U(VI)). One species whose vegetative cells are capable of U(VI) reduction is C. acetobutylicum, although little information is available on the mechanism underlying this process. In this thesis, the potential involvement of the spores of C. acetobutylicum in U(VI) reduction is probed. It was found that the spores of this species can reduce the radionuclide, and that to do so they require the presence of an unidentified soluble compound released by growing cells and H2 as the electron donor. The involvement of spores in metal reduction is very interesting because generally spores are considered not to interact directly with the surrounding environment. In fact, spores are dormant, thus metabolically inactive, cells. Spore formation is a cell differentiation process activated in response to environmental stress and conditions that are hostile to vegetative growth. Sporulation has been extensively studied and characterized in Bacillus subtilis, for which a model has been developed. Some information is also available on sporulation in the clostridia. In this thesis, a genomic investigation of sporulation in the Desulfotomaculum genus was undertaken. The B. subtilis model was used as a reference and the information on Clostridium as a comparison. Significant similarities were found in the core regulatory process across genera, while some differences were identified in the initiation of sporulation. Substantial differences were highlighted in the spore structural proteins.

EPFL2014

Utilization of silicate minerals for pH control during in situ bioremediation of chlorinated solvent

David Andrew Barry, Alessandro Brovelli, Christof Holliger, Elsa Marina Lacroix

Chloroethenes such as tetrachloroethene (PCE) and trichloroethene (TCE) are among the most prevalent contaminants in groundwater due to their extensive use in industrial processes. In situ bioremediation (ISB) is an attractive technology for removal of these compounds. It relies on an anaerobic process in which specialized bacteria obtain energy for growth using chloroethenes as an electron acceptor via organohalide respiration. Engineered bioremediation is achieved by stimulating these microorganisms through the addition of electron donor in the subsurface. This technology has been widely used for bioremediation of chloroethene plumes and recent studies have indicated promising results for bioremediation of chlorinated solvent source zones. However, application of source zone ISB is still a significant technical challenge. One of the main issues is the groundwater acidification due to organohalide respiration and fermentation processes, which can inhibit the activity of dehalogenating micro-organisms. The main objective of this work was to develop an efficient pH control strategy for chloroethene ISB by using the acid neutralizing potential of silicate minerals. To do so, modeling and experimental approaches were combined. A geochemical model, implemented within PHREEQC, was developed to select appropriate buffer candidates and to help determine main parameters influencing mineral buffering capacity. The model included chloroethene microbial degradation kinetics, mineral dissolution kinetics and chemical speciation. Second, anaerobic microcosm experiments were performed to determine the influence of pH on dehalogenation. These microcosms were inoculated with enriched consortia of dehalogenating bacteria and fed with PCE and hydrogen. Another set of microcosm experiments was carried out to compare the buffering capacity of ten silicate minerals and to investigate interactions between minerals and dehalogenating bacteria, e.g., the potential inhibitory effect of minerals on the dehalogenating activity. These microcosms were amended with 5 mmol l-1 of PCE and 4 g l-1 of mineral with grain sizes between 50 and 100 μm. The cultivation medium was modified such that the silicate mineral powder was the sole pH buffer present. Chloroethenes, pH and dissolved cation measurements were conducted to determine the system efficiency. Abiotic dissolution experiments were also performed to determine mineral dissolution rates in the absence of bacteria. The model confirmed that the efficiency of the system is dependent mainly on mineral dissolution kinetic constants, equilibrium constants and reactive surface area. The geochemical model and literature parameter data were used to pre-select minerals with a buffering capacity sufficient to counterbalance acidity produced by dehalogenating bacteria at a rate of 4 mmol l-1.d-1of chloride. Of the 31 silicate minerals for which there were published kinetic data, 10 were identified as suitable candidates. The inhibitory pH for the dehalogenating consortia was found to vary between 5 and 6. The last steps of the dechlorination from DCE to ethene were more sensitive to pH than the first steps from PCE to DCE, as has been noted in other dechlorination studies. Results of microcosm experiments with silicate minerals demonstrated that, under the selected conditions, the pH control behavior and the impact on bacterial activity exhibited strong variations depending on the mineral. Of the ten minerals tested experimentally, three (olivine, fayalite and diopside) maintained the pH in the appropriate range, i.e., between 5.5 and 6.5 and led to complete transformation of PCE. For the other minerals tested, either the acid neutralization capacity was insufficient due to slow dissolution kinetics (glaucophane and staurolite) or dechlorination activity was inhibited by (unmeasured) compounds released during mineral dissolution. Both modeling and experimental results demonstrated the feasibility of using selected silicate minerals as a buffering agent during ISB. However, the experimental results also revealed a mineral-induced potential inhibitory effect that should be investigated prior to application at a contaminated site.

2011