Fe(III) reduction during pyruvate fermentation by Desulfotomaculum reducens strain MI-1

Desulfotomaculum reducens MI-1 is a Gram-positive, sulfate-reducing bacterium also capable of reducing several metals, among which is Fe(III). Very limited knowledge is available on the potential mechanism(s) of metal reduction among Gram-positive bacteria, despite their preponderance in the microbial communities that inhabit some inhospitable environments (e.g., thermal or hyperthermal ecosystems, extreme pH or salinity environments, heavy metal or radionuclide contaminated sediments). Here, we show that in the presence of pyruvate, this micro-organism is capable of reducing both soluble Fe(III)-citrate and solid-phase hydrous ferric oxide, although growth is sustained by pyruvate fermentation rather than Fe(III) respiration. Despite the fact that Fe(III) reduction does not support direct energy conservation, D.reducens uses it as a complementary means of discarding excess reducing equivalent after H-2 accumulation in the culture headspace renders proton reduction unfavorable. Thus, Fe(III) reduction permits the oxidation of greater amounts of pyruvate than fermentation alone. Fe(III) reduction by D.reducens is mediated by a soluble electron carrier, most likely riboflavin. Additionally, an intracellular electron storage molecule acts as a capacitor and accumulates electrons during pyruvate oxidation for slow release to Fe(III). The reductase responsible for the transfer of electrons from the capacitor to the soluble carrier has not been identified, but data presented here argue against the involvement of c-type cytochromes.

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

Assessing the Bacterial Ecology of Organohalide Respiration for the Design of Bioremediation Strategies

Noam Shani

Groundwater is essential for human activities and is sometimes referred to as a non-renewable resource in the same way as oil and gas. During the past century, precious groundwater reserves worldwide have been threatened by anthropogenic release of chemical compounds. Among these, chlorinated ethenes (CEs) such as tetrachloroethene (PCE) and trichloroethene (TCE) belong to the most common class of groundwater contaminants. In oxic conditions, PCE and TCE are recalcitrant to any form of degradation and constitute a long-term source of groundwater contamination. It has been reported that CEs can be degraded biologically in anoxic conditions, serving as electron acceptors of an anaerobic respiratory process called organohalide respiration (OHR). In this process, PCE is sequentially reduced to TCE, cis-dichloroethene (cDCE), vinyl chloride (VC), and finally to harmless ethene (Eth). Engineering strategies based on reductive dechlorination, such as monitored natural attenuation (MNA), have been designed for the bioremediation of aquifers contaminated with CEs. However, stalling of the sequence of dechlorination has often been observed, resulting from incomplete or impeded biodegradation of the highly toxic daughter molecules (cDCE and VC) and leading to their accumulation in situ. It was shown in laboratory experiments that OHR of CEs is more efficient in mixed cultures and that organohalide-respiring bacteria (OHRB) live in association with other microorganisms in microbial consortia. In the aquifer ecosystem, OHRB rely on complex interactions between members of bacterial communities for their electron donors and carbon supplies. On the other hand, they are in competition for these resources with other terminal electron-accepting processes (TEAPs). These complex interactions together with the intrinsic structural heterogeneity of aquifers make it difficult to understand the reasons for lower CEs accumulation, to predict the fate of OHR, and to design bioremediation strategies. This thesis aimed at elucidating the reasons for lower CEs accumulation in situ. An ecological approach considering the aquifer ecosystem in its whole complexity was developed and applied to the specific cases of two contaminated sites showing accumulation of lower CEs. The proposed methodology relies on the precise depiction of both aquifer habitat and microbial communities interacting therein. However, the potential impact of the first technical step, namely the impact of the pumping parameters on groundwater samples used for the description of the bacterial communities, was not known at the start of this thesis work. Results of investigations addressing this topic showed that parameters related to the tubing characteristics did not impact the apparent bacterial community structures (BCS) in a laboratory experiment. However, the study revealed a significant impact of the pumping flow rate on apparent BCS extracted from the aquifer. Terminal-restriction fragment length polymorphism (T-RFLP), coupled with an appropriate groundwater sampling strategy, enabled an accurate profiling of the BCS. However no indication was provided concerning the identities of the community members. A bioinformatics tool called PyroTRF-ID was developed to overcome this obstacle. The software enabled affiliating terminal-restriction fragments (T-RFs) to precise phylotypes by coupling T-RFLP and pyrosequencing data. An additional function enables in silico assessment of restriction enzymes for designing appropriate T-RFLP procedures. The developed methodology was applied all along the thesis work. Optimized tools and procedures were used to investigate the reasons for the accumulation of VC, and to a lesser extent cDCE, in the first aquifer showing relatively homogeneous lithological composition and slow groundwater fluxes. Statistical findings indicated that VC reduction was outcompeted by Fe(III) reduction in some sections of the aquifer. T-RFs showing significant correlations with VC reduction variables were identified by sequencing and with PyroTRF-ID. Results showed sequences closely affiliated to uncultured bacteria of the "Lahn Cluster" (LC) within the class Dehalococcoidetes, previously reported as PCE-to-cDCE dechlorinating microorganisms, and to the genus "Dehalococcoides". According to present knowledge, only members of this genus are able to reduce cDCE and VC. A major T-RF negatively correlated with the LC T-RF affiliated to the genus Rhodoferax, containing iron-reducing bacteria (IRB). Furthermore, indications were obtained of a mutualistic interaction between IRB and sulfate-reducing bacteria, potentially playing an important role by reducing the Fe(III) contents locally. The importance of the aquifer hydrogeological structure and functioning was exemplified in the study of the second aquifer ecosystem, where discrepancies in the fate of OHR were observed. In the immediate vicinity of the source zone, complete OHR was occurring, with production of Eth, whereas cDCE was accumulating further downstream, in apparent similar redox conditions. The profiling and metagenomic techniques revealed drastically different bacterial communities in the zones displaying different OHR fate. The source zone displayed bacterial populations typically found in highly reduced environments and involved in OHR, such as Dehalococcoides sp. In contrast, the downstream zone showing an accumulation of cDCE displayed bacterial populations typically found in more oxidized environments, such as aerobic bacteria, nitrifiers, and denitrifiers. In this zone, the conditions were probably slightly oxidized periodically by the combination of aquifer recharge, a specific lithological configuration, and lower organic matter content. Finally, and based on these considerations, a methodology for the investigation of the reasons for lower CEs accumulation in contaminated aquifers is proposed and discussed. This methodology follows on a multidisciplinary ecological approach for the description of the functioning of the aquifer ecosystem, and enables formulating scenarii of the reasons for lower CEs accumulation.

EPFL2012

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