Glycyl Radical Enzymes and Sulfonate Metabolism in the Microbiome.

Sulfonates include diverse natural products and anthropogenic chemicals and are widespread in the environment. Many bacteria can degrade sulfonates and obtain sulfur, carbon, and energy for growth, playing important roles in the biogeochemical sulfur cycle. Cleavage of the inert sulfonate C-S bond involves a variety of enzymes, cofactors, and oxygen-dependent and oxygen-independent catalytic mechanisms. Sulfonate degradation by strictly anaerobic bacteria was recently found to involve C-S bond cleavage through O2-sensitive free radical chemistry, catalyzed by glycyl radical enzymes (GREs). The associated discoveries of new enzymes and metabolic pathways for sulfonate metabolism in diverse anaerobic bacteria have enriched our understanding of sulfonate chemistry in the anaerobic biosphere. An anaerobic environment of particular interest is the human gut microbiome, where sulfonate degradation by sulfate- and sulfite-reducing bacteria (SSRB) produces H2S, a process linked to certain chronic diseases and conditions.

Bacterial cysteate dissimilatory pathway involves a racemase and d-cysteate sulfo-lyase.

The sulfite-reducing bacterium Bilophila wadsworthia, a common human intestinal pathobiont, is unique in its ability to metabolize a wide variety of sulfonates to generate sulfite as a terminal electron acceptor (TEA). The resulting formation of H2S is implicated in inflammation and colon cancer. l-cysteate, an oxidation product of l-cysteine, is among the sulfonates metabolized by B. wadsworthia, although the enzymes involved remain unknown. Here we report a pathway for l-cysteate dissimilation in B. wadsworthia RZATAU, involving isomerization of l-cysteate to d-cysteate by a cysteate racemase (BwCuyB), followed by cleavage into pyruvate, ammonia and sulfite by a d-cysteate sulfo-lyase (BwCuyA). The strong selectivity of BwCuyA for d-cysteate over l-cysteate was rationalized by protein structural modeling. A homolog of BwCuyA in the marine bacterium Silicibacter pomeroyi (SpCuyA) was previously reported to be a l-cysteate sulfo-lyase, but our experiments confirm that SpCuyA too displays a strong selectivity for d-cysteate. Growth of B. wadsworthia with cysteate as the electron acceptor is accompanied by production of H2S and induction of BwCuyA. Close homologs of BwCuyA and BwCuyB are present in diverse bacteria, including many sulfate- and sulfite-reducing bacteria, suggesting their involvement in cysteate degradation in different biological environments.

Isethionate is an intermediate in the degradation of sulfoacetate by the human gut pathobiont Bilophila wadsworthia.

The obligately anaerobic sulfite-reducing bacterium Bilophila wadsworthia is a common human pathobiont inhabiting the distal intestinal tract. It has a unique ability to utilize a diverse range of food- and host-derived sulfonates to generate sulfite as a terminal electron acceptor (TEA) for anaerobic respiration, converting the sulfonate sulfur to H2S, implicated in inflammatory conditions and colon cancer. The biochemical pathways involved in the metabolism of the C2 sulfonates isethionate and taurine by B. wadsworthia were recently reported. However, its mechanism for metabolizing sulfoacetate, another prevalent C2 sulfonate, remained unknown. Here, we report bioinformatics investigations and in vitro biochemical assays that uncover the molecular basis for the utilization of sulfoacetate as a source of TEA (STEA) for B. wadsworthia, involving conversion to sulfoacetyl-CoA by an ADP-forming sulfoacetate-CoA ligase (SauCD), and stepwise reduction to isethionate by NAD(P)H-dependent enzymes sulfoacetaldehyde dehydrogenase (SauS) and sulfoacetaldehyde reductase (TauF). Isethionate is then cleaved by the O2-sensitive isethionate sulfolyase (IseG), releasing sulfite for dissimilatory reduction to H2S. Sulfoacetate in different environments originates from anthropogenic sources such as detergents, and natural sources such as bacterial metabolism of the highly abundant organosulfonates sulfoquinovose and taurine. Identification of enzymes for anaerobic degradation of this relatively inert and electron-deficient C2 sulfonate provides further insights into sulfur recycling in the anaerobic biosphere, including the human gut microbiome.

Redox Properties of Solid Phase Electron Acceptors Affect Anaerobic Microbial Respiration under Oxygen-Limited Conditions in Floodplain Soils.

Mountain floodplain soils often show spatiotemporal variations in redox conditions that arise due to changing hydrology and resulting biogeochemistry. Under oxygen-depleted conditions, solid phase terminal electron acceptors (TEAs) can be used in anaerobic respiration. However, it remains unclear to what degree the redox properties of solid phases limit respiration rates and hence organic matter degradation. Here, we assess such limitations in soils collected across a gradient in native redox states from the Slate River floodplain (Colorado, U.S.A.). We incubated soils under anoxic conditions and quantified CO2 production and microbial Fe(III) reduction, the main microbial metabolic pathway, as well as the reactivity of whole-soil solid phase TEAs toward mediated electrochemical reduction. Fe(III) reduction occurred together with CO2 production in native oxic soils, while neither Fe(II) nor CO2 production was observed in native anoxic soils. Initial CO2 production rates increased with increasing TEA redox reactivity toward mediated electrochemical reduction across all soil depths. Low TEA redox reactivity appears to be caused by elevated Fe(II) concentrations rather than crystallinity of Fe(III) phases. Our findings illustrate that the buildup of Fe(II) in systems with long residence times limits the thermodynamic viability of dissimilatory Fe(III) reduction and thereby limits the mineralization of organic carbon.

Anaerobic degradation of xenobiotic organic contaminants (XOCs): The role of electron flow and potential enhancing strategies.

In groundwater, deep soil layer, sediment, the widespread of xenobiotic organic contaminants (XOCs) have been leading to the concern of human health and eco-environment safety, which calls for a better understanding on the fate and remediation of XOCs in anoxic matrices. In the absence of oxygen, bacteria utilize various oxidized substances, e.g. nitrate, sulphate, metallic (hydr)oxides, humic substance, as terminal electron acceptors (TEAs) to fuel anaerobic XOCs degradation. Although there have been increasing anaerobic biodegradation studies focusing on species identification, degrading pathways, community dynamics, systematic reviews on the underlying mechanism of anaerobic contaminants removal from the perspective of electron flow are limited. In this review, we provide the insight on anaerobic biodegradation from electrons aspect - electron production, transport, and consumption. The mechanism of the coupling between TEAs reduction and pollutants degradation is deconstructed in the level of community, pure culture, and cellular biochemistry. Hereby, relevant strategies to promote anaerobic biodegradation are proposed for guiding to an efficient XOCs bioremediation.

How does electron transfer occur in microbial fuel cells?

Microbial fuel cells (MFCs) have emerged as a promising technology for sustainable wastewater treatment coupled with electricity generation. A MFC is a device that uses microbes as catalysts to convert chemical energy present in biomass into electrical energy. Among the various mechanisms that drive the operation of a MFC, extracellular electron transfer (EET) to the anode is one of the most important. Exoelectrogenic bacteria can natively transfer electrons to a conducting surface like the anode. The mechanisms employed for electron transfer can either be direct transfer via conductive pili or nanowires, or mediated transfer that involves either naturally secreted redox mediators like flavins and pyocyanins or artificially added mediators like methylene blue and neutral red. EET is a mechanism wherein microorganisms extract energy for growth and maintenance from their surroundings and transfer the resulting electrons to the anode to generate current. The efficiency of these electron transfer mechanisms is dependent not only on the redox potentials of the species involved, but also on microbial oxidative metabolism that liberates electrons. Attempts at understanding the electron transfer mechanisms will boost efforts in giving rise to practical applications. This article covers the various electron transfer mechanisms involved between microbes and electrodes in microbial fuel cells and their applications.

Biodegradation of petroleum hydrocarbons and changes in microbial community structure in sediment under nitrate-, ferric-, sulfate-reducing and methanogenic conditions.

In the present study, the biodegradation behaviors of petroleum hydrocarbons under various reducing conditions were investigated. n-Alkanes and polycyclic aromatic hydrocarbons (PAHs) were degraded with NO3-, Fe3+, SO42-, or HCO3- as terminal electron acceptors (TEAs), which link to four typical reducing conditions (i.e., nitrate-reducing, ferric-reducing, sulfate-reducing and methanogenic conditions, respectively) in sediment. The fastest degradation rates were achieved under sulfate-reducing conditions with half-lives of 49.51 days for n-alkanes and 58.74 days for PAHs. For short-chain n-alkanes and low-molecular weight (LMW) PAHs, relatively higher removal efficiencies were achieved under nitrate- and ferric-reducing conditions. The degradation of long-chain n-alkanes and high-molecular weight (HMW) PAHs coupled to methanogenesis was the most favored as compared with other reducing conditions. Carboxylation was found to be the principle mechanism for regulating n-alkane degradation coupled to denitrification, while the activation of n-alkanes by the addition of fumarate was the principle mechanism for the n-alkane degradation under sulfate-reducing conditions. The anaerobic metabolism of n-alkanes may not proceed via fumarate addition or carboxylation under ferric-reducing and methanogenic conditions. Illumina HiSeq sequencing revealed dissimilar structures of the microbial communities under various reducing conditions. It is hypothesized that the utilization of different TEAs for n-alkane and PAH degradation resulted in distinct microbial community structures, which were highly correlated with the varied degradation behaviors of petroleum hydrocarbons in sediment. The current results may provide reference value on better understanding the biodegradation behaviors of n-alkanes and PAHs in association with the induced microbial communities in sedimentary environments under the four typical reducing conditions.

Biofilm Formation by Uropathogenic Escherichia coli Is Favored under Oxygen Conditions That Mimic the Bladder Environment.

One of the most common urologic problems afflicting millions of people worldwide is urinary tract infection (UTI). The severity of UTIs ranges from asymptomatic bacteriuria to acute cystitis, and in severe cases, pyelonephritis and urosepsis. The primary cause of UTIs is uropathogenic Escherichia coli (UPEC), for which current antibiotic therapies often fail. UPEC forms multicellular communities known as biofilms on urinary catheters, as well as on and within bladder epithelial cells. Biofilm formation protects UPEC from environmental conditions, antimicrobial therapy, and the host immune system. Previous studies have investigated UPEC biofilm formation in aerobic conditions (21% oxygen); however, urine oxygen tension is reduced (4-6%), and urine contains molecules that can be used by UPEC as alternative terminal electron acceptors (ATEAs) for respiration. This study was designed to determine whether these different terminal electron acceptors utilized by E. coli influence biofilm formation. A panel of 50 urine-associated E. coli isolates was tested for the ability to form biofilm under anaerobic conditions and in the presence of ATEAs. Biofilm production was reduced under all tested sub-atmospheric levels of oxygen, with the notable exception of 4% oxygen, the reported concentration of oxygen within the bladder.

Effects of terminal electron acceptors on the biodegradation of waste motor oil using Chlorella vulgaris-Rhodococcus erythropolis consortia: Kinetic and thermodynamic windows of opportunity analysis.

The Chlorella vulgaris-Rhodococcus erythropolis consortia was constructed for the biodegradation of waste motor oil (WMO), combined with thermodynamic calculations and stoichiometric analyses. The microalgae-bacteria consortium was constructed as C. vulgaris: R. erythropolis = 1:1 (biomass, cell/mL), pH = 7, 3 g/L WMO. Under the same condition, the terminal electron acceptors (TEAs) play a crucial role in the WMO biodegradation, which follows Fe3+ >SO42- > none. The biodegradation of WMO fitted well with the first-order kinetic model under experimental temperatures with different TEAs (R2 >0.98). The WMO biodegradation efficiency reached 99.2 % and 97.1 % with Fe3+ and SO42-as TEAs at 37 °C, respectively. Thermodynamic methanogenesis opportunity windows with Fe3+ as TEA are 2.72 times fold as large as those with SO42-. Microorganism metabolism equations demonstrated the viability of anabolism and catabolism on WMO. This work lays the groundwork for the implementation of WMO wastewater bioremediation and supports research into the biochemical process of WMO biotransformation.

Anaerobic ammonium oxidation (anammox) promoted by pyrogenic biochar: Deciphering the interaction with extracellular polymeric substances (EPS).

Efficient biological nitrogen removal (BNR) by anaerobic ammonium oxidation (anammox) can be achieved with presence of redox-active pyrogenic biochar that potentially acting as an insoluble electron acceptor. Anammox bacteria and other symbiotic consortia are surrounded by extracellular polymeric substances (EPS) forming aggregate architecture, which also contains electrochemical-active biomolecules such as aromatic proteins and humic substances. Therefore, understanding the role of EPS is necessary in biochar-promoting anammox process. Herein, we investigated the influence of biochar with granular-sized (GP) and micrometer-sized (MP) particle sizes on microbiology and characteristics of EPS in anammox sludge. Addition of GP and MP biochar not only improved the BNR efficiency by 17.5% and 34.6%, respectively, but also increased the relative abundance of Candidatus Brocadia. The bulk and bound EPS contents substantially decreased in biochar-amended groups, while more slime EPS was produced. Spectroscopic (FTIR, Raman, and circular dichroism) and electrochemical (voltammetry and impedance spectrum) analyses revealed that biochar addition enhanced the structural integrity and electron-transfer capability of anammox sludge. EPS depletion led to a steep decrease in BNR efficiency (21.5% vs 83.0% with EPS-retained sludge), whereas it resumed to 42.1% in the presence of MP biochar. Electron transport system activity data showed that biochar replenished the loss of anaerobic respiration metabolism due to EPS depletion. In summary, these results suggested that EPS possibly work as transient mediator for shuttling electrons from ammonium oxidation to soluble (nitrite) and insoluble electron acceptors (redox-active biochar).

Anodic electro-fermentation: Empowering anaerobic production processes via anodic respiration.

In nature as well as in industrial microbiology, all microorganisms need to achieve redox balance. Their redox state and energy conservation highly depend on the availability of a terminal electron acceptor, for example oxygen in aerobic production processes. Under anaerobic conditions in the absence of an electron acceptor, redox balance is achieved via the production of reduced carbon-compounds (fermentation). An alternative strategy to artificially stabilize microbial redox and energy state is the use of anodic electro-fermentation (AEF). This emerging biotechnology empowers respiration under anaerobic conditions using the anode of a bioelectrochemical system as an undepletable terminal electron acceptor. Electrochemical control of redox metabolism and energy conservation via AEF can steer the carbon metabolism towards a product of interest and avoid the need for continuous and cost-inefficient supply of oxygen as well as the production of mixed reduced by-products, as is the case in aerobic production and fermentation processes, respectively. The great challenge for AEF is to establish efficient extracellular electron transfer (EET) from the microbe to the anode and link it to central carbon metabolism to enhance the synthesis of a target product. This article reviews the advantages and challenges of AEF, EET mechanisms, microbial energy gain, and discusses the rational choice of substrate-product couple as well as the choice of microbial catalyst. Besides, it discusses the potential of the industrial model-organism Bacillus subtilis as a promising candidate for AEF, which has not been yet considered for such an application. This prospective review contributes to a better understanding of how industrial microbiology can benefit from AEF and analyses key-factors required to successfully implement AEF processes. Overall, this work aims to advance the young research field especially by critically revisiting the fundamental aspects of AEF.

Effect of humic acids on batch anaerobic digestion of excess sludge.

Anaerobic digestion (AD) is a sustainable pathway towards recovering chemical energy from excess sludge, and humic substances (HSs) contained in sludge can inhibit energy (methane/CH4) conversion efficiency. This study aims to investigate the impact of humic acids (HA) on the various processes in a batch anaerobic digestion process. For this purpose, "clean" sludge was cultivated in a laboratory to avoid HSs presence. The cultivated sludge was used in a series of batch experiments, with humic acids added at different levels. A complete AD test, as well as three sub-phase tests (hydrolytic phase; acidogenic phase; methanogenic phase) was performed and analyzed with and without HA dosing. In the single-phase AD system, dosing with HA inhibited the methanogenic efficiency by 35.1% at HA:VSS = 15%. However, the effects of HA on the three sub-phases revealed something very different. HA inhibited hydrolytic efficiency by 38.2%, promoted acidogenic efficiency by 101.5%, and finally inhibited methanogenic efficiency by 52.2%. The combined efficiency of the three sub-phases without HA dosing is calculated at 15.7%; and with HA dosing (HA:VSS = 15%) at 10.2%. Overall, the combined inhibition efficiency of the three sub-phases is equal to 35.0%, which is almost identical (35.1%) to the result observed in the single-phase AD process. The possible mechanisms behind the phenomena were analyzed and summarized in the context.

Assessment of the performance of SMFCs in the bioremediation of PAHs in contaminated marine sediments under different redox conditions and analysis of the associated microbial communities.

The biodegradation of naphthalene, 2-methylnaphthalene and phenanthrene was evaluated in marine sediment microbial fuel cells (SMFCs) under different biodegradation conditions, including sulfate reduction as a major biodegradation pathway, employment of anode as terminal electron acceptor (TEA) under inhibited sulfate reducing bacteria activity, and combined sulfate and anode usage as electron acceptors. A significant removal of naphthalene and 2-methylnaphthalene was observed at early stages of incubation in all treatments and was attributed to their high volatility. In the case of phenanthrene, a significant removal (93.83±1.68%) was measured in the closed circuit SMFCs with the anode acting as the main TEA and under combined anode and sulfate reduction conditions (88.51±1.3%). A much lower removal (40.37±3.24%) was achieved in the open circuit SMFCs operating with sulfate reduction as a major biodegradation pathway. Analysis of the anodic bacterial community using 16S rRNA gene pyrosequencing revealed the enrichment of genera with potential exoelectrogenic capability, namely Geoalkalibacter and Desulfuromonas, on the anode of the closed circuit SMFCs under inhibited SRB activity, while they were not detected on the anode of open circuit SMFCs. These results demonstrate the role of the anode in enhancing PAHs biodegradation in contaminated marine sediments and suggest a higher system efficiency in the absence of competition between microbial redox processes (under SRB inhibition), namely due to the anode enrichment with exoelectrogenic bacteria, which is a more energetically favorable mechanism for PAHs oxidation than sulfate.

Influences of groundwater extraction on the distribution of dissolved As in shallow aquifers of West Bengal, India.

Here we report temporal changes of As concentrations in shallow groundwater of the Bengal Delta Plain (BDP). Observed fluctuations are primarily induced by seasonally occurring groundwater movement, but can also be connected to anthropogenic groundwater extraction. Between December 2009 and July 2010, pronounced variations in the groundwater hydrochemistry were recorded in groundwater samples of a shallow monitoring well tapping the aquifer in 22-25 m depth, where Astot concentrations increased within weeks from 100 to 315 µg L(-1). These trends are attributed to a vertically shift of the hydrochemically stratified water column at the beginning of the monsoon season. This naturally occurring effect can be additionally superimposed by groundwater extraction, as demonstrated on a local scale by an in situ experiment simulating extensive groundwater withdrawal during the dry post-monsoon season. Results of this experiment suggest that groundwater extraction promoted an enduring change within the distribution of dissolved As in the local aquifer. Presented outcomes contribute to the discussion of anthropogenic pumping influences that endanger the limited and yet arsenic-free groundwater resources of the BDP.

Enhanced performance of sulfate reducing bacteria based biocathode using stainless steel mesh on activated carbon fabric electrode.

An anoxic biocathode was developed using sulfate-reducing bacteria (SRB) consortium on activated carbon fabric (ACF) and the effect of stainless steel (SS) mesh as additional current collector was investigated. Improved performance of biocathode was observed with SS mesh leading to nearly five folds increase in power density (from 4.79 to 23.11 mW/m(2)) and threefolds increase in current density (from 75 to 250 mA/m(2)). Enhanced redox currents and lower Tafel slopes observed from cyclic voltammograms of ACF with SS mesh indicated the positive role of uniform electron collecting points. Differential pulse voltammetry technique was employed as an additional tool to assess the redox carriers involved in bioelectrochemical reactions. SRB biocathode was also tested for reduction of volatile fatty acids (VFA) present in the fermentation effluent stream and the results indicated the possibility of integration of this system with anaerobic fermentation for efficient product recovery.

The small protein CydX is required for function of cytochrome bd oxidase in Brucella abortus.

A large number of hypothetical genes potentially encoding small proteins of unknown function are annotated in the Brucella abortus genome. Individual deletion of 30 of these genes identified four mutants, in BAB1_0355, BAB2_0726, BAB2_0470, and BAB2_0450 that were highly attenuated for infection. BAB2_0726, an YbgT-family protein located at the 3' end of the cydAB genes encoding cytochrome bd ubiquinal oxidase, was designated cydX. A B. abortus cydX mutant lacked cytochrome bd oxidase activity, as shown by increased sensitivity to H(2)O(2), decreased acid tolerance and increased resistance to killing by respiratory inhibitors. The C terminus, but not the N terminus, of CydX was located in the periplasm, suggesting that CydX is an integral cytoplasmic membrane protein. Phenotypic analysis of the cydX mutant, therefore, suggested that CydX is required for full function of cytochrome bd oxidase, possibly via regulation of its assembly or activity.