Analog Anthraquinone-2,6-disulfonate Research Articles

Metal Transformation by a Novel Pelosinus Isolate From a Subsurface Environment.

The capability of microorganisms to alter metal speciation offers potential for the development of new strategies for immobilization of toxic metals in the environment. A metal-reducing microbe, “Pelosinus lilae” strain UFO1, was isolated under strictly anaerobic conditions from an Fe(III)-reducing enrichment established with uncontaminated soil from the Department of Energy Oak Ridge Field Research Center, Tennessee. “P. lilae” UFO1 is a rod-shaped, spore-forming, and Gram-variable anaerobe with a fermentative metabolism. It is capable of reducing the humic acid analog anthraquinone-2,6-disulfonate (AQDS) using a variety of fermentable substrates and H2. Reduction of Fe(III)-nitrilotriacetic acid occurred in the presence of lactate as carbon and electron donor. Ferrihydrite was not reduced in the absence of AQDS. Nearly complete reduction of 1, 3, and 5 ppm Cr(VI) occurred within 24 h in suspensions containing 108 cells mL−1 when provided with 10 mM lactate; when 1 mM AQDS was added, 3 and 5 ppm Cr(VI) were reduced to 0.1 ppm within 2 h. Strain UFO1 is a novel species within the bacterial genus Pelosinus, having 98.16% 16S rRNA gene sequence similarity with the most closely related described species, Pelosinus fermentans R7T. The G+C content of the genomic DNA was 38 mol%, and DNA-DNA hybridization of “P. lilae” UFO1 against P. fermentans R7T indicated an average 16.8% DNA-DNA similarity. The unique phylogenetic, physiologic, and metal-transforming characteristics of “P. lilae” UFO1 reveal it is a novel isolate of the described genus Pelosinus.

Peatland types influence the inhibitory effects of a humic substance analog on methane production

Methane (CH4) production is often impeded in many northern peatland soils, even though more thermodynamically favorable inorganic terminal electron acceptors (TEAs) used in anaerobic respiration are often present in low concentrations in these soils. Recent studies suggest that humic substances in wetland soils can be utilized as organic TEAs for anaerobic respiration and may directly inhibit CH4 production. Here we utilize the humic analog anthraquinone-2, 6-disulfonate (AQDS) to explore the importance of humic substances, and their effects on the temperature sensitivity of anaerobic decomposition, in two peatland soils. In a bog peat, AQDS was not initially utilized as a TEA, but greatly inhibited the fermentative production of acetate by >98%, carbon dioxide by >49%, hydrogen by >90%, and CH4 production by 86%. When added together with glucose, <47% of added AQDS was reduced after a lag period of 5 to 10days. In contrast, no inhibitory effect of AQDS on fermentation was found in a fen peat, and AQDS was readily reduced. The addition of glucose and AQDS to both bog and fen peats caused complicated temporal dynamics in the temperature sensitivity of CH4 production, reflecting temporal changes in the temperature responses of other decomposition processes with effects on methanogenesis. Our results show that the inhibitory effects of a humic analog on CH4 production depend on peatland type, acting primarily as a toxic compound in bog soils and as a competitive TEA in fen soils. The results also suggest that the high concentrations of humic substances in northern peatlands may impact the temperature sensitivity of soil carbon cycling in these ecosystems.

Humic analog AQDS can act as a selective inhibitor to enable anoxygenic photosynthetic bacteria to outcompete sulfate‐reducing bacteria under microaerobic conditions

ABSTRACTBACKGROUNDThe anoxygenic photosynthetic bacteria (APB) process is considered the most promising candidate for cost‐effective wastewater treatment and resource recovery. One major problem in the APB process with sulfate‐containing wastewater is the overgrowth of sulfate‐reducing bacteria. In this study, the humic analog anthraquinone‐2,6‐disulfonate (AQDS) was used to selectively inhibit sulfate‐reducing bacteria (SRB) in an SRB and APB coculture.RESULTSThe addition of 1.0 mmol L−1 AQDS to the SRB and APB coculture resulted in significant decreases in the amount of SRB and the sulfide concentration, whereas the number of APB increased from 3.7 × 103 to 3.1 × 108 CFU mL−1. A high concentration of AQDS negatively affected the growth of both SRB and APB but more strongly inhibited the growth of SRB. The inhibition of SRB by AQDS relies on an electron donor and is enhanced by illumination. AQDS treatment at 1.0 mmol L−1 induced significant membrane depolarization and superoxide production in SRB cells but not in APB cells, and this may be responsible for the selective inhibition of SRB in the coculture.CONCLUSIONAt appropriate concentrations, AQDS can decrease the concentration of sulfide and induce oxidative stress for SRB, effectively allowing APB to out‐compete SRB under microaerobic conditions. © 2015 Society of Chemical Industry

Molecular interaction studies revealed the bifunctional behavior of triheme cytochrome PpcA from Geobacter sulfurreducens toward the redox active analog of humic substances

Humic substances (HS) constitute a significant fraction of natural organic matter in terrestrial and aquatic environments and can act as terminal electron acceptors in anaerobic microbial respiration. Geobacter sulfurreducens has a remarkable respiratory versatility and can utilize the HS analog anthraquinone-2,6-disulfonate (AQDS) as a terminal electron acceptor or its reduced form (AH2QDS) as an electron donor. Previous studies set the triheme cytochrome PpcA as a key component for HS respiration in G. sulfurreducens, but the process is far from fully understood. In this work, NMR chemical shift perturbation measurements were used to map the interaction region between PpcA and AH2QDS, and to measure their binding affinity. The results showed that the AH2QDS binds reversibly to the more solvent exposed edge of PpcA heme IV. The NMR and visible spectroscopies coupled to redox measurements were used to determine the thermodynamic parameters of the PpcA:quinol complex. The higher reduction potential of heme IV (−127mV) compared to that of AH2QDS (−184mV) explains why the electron transfer is more favorable in the case of reduction of the cytochrome by the quinol. The clear evidence obtained for the formation of an electron transfer complex between AH2QDS and PpcA, combined with the fact that the protein also formed a redox complex with AQDS, revealed for the first time the bifunctional behavior of PpcA toward an analog of the HS. Such behavior might confer selective advantage to G. sulfurreducens, which can utilize the HS in any redox state available in the environment for its metabolic needs.

Syntrophic growth via quinone-mediated interspecies electron transfer.

The mechanisms by which microbial species exchange electrons are of interest because interspecies electron transfer can expand the metabolic capabilities of microbial communities. Previous studies with the humic substance analog anthraquinone-2,6-disulfonate (AQDS) suggested that quinone-mediated interspecies electron transfer (QUIET) is feasible, but it was not determined if sufficient energy is available from QUIET to support the growth of both species. Furthermore, there have been no previous studies on the mechanisms for the oxidation of anthrahydroquinone-2,6-disulfonate (AHQDS). A co-culture of Geobacter metallireducens and G. sulfurreducens metabolized ethanol with the reduction of fumarate much faster in the presence of AQDS, and there was an increase in cell protein. G. sulfurreducens was more abundant, consistent with G. sulfurreducens obtaining electrons from acetate that G. metallireducens produced from ethanol, as well as from AHQDS. Co-cultures initiated with a citrate synthase-deficient strain of G. sulfurreducens that was unable to use acetate as an electron donor also metabolized ethanol with the reduction of fumarate and cell growth, but acetate accumulated over time. G. sulfurreducens and G. metallireducens were equally abundant in these co-cultures reflecting the inability of the citrate synthase-deficient strain of G. sulfurreducens to metabolize acetate. Evaluation of the mechanisms by which G. sulfurreducens accepts electrons from AHQDS demonstrated that a strain deficient in outer-surface c-type cytochromes that are required for AQDS reduction was as effective at QUIET as the wild-type strain. Deletion of additional genes previously implicated in extracellular electron transfer also had no impact on QUIET. These results demonstrate that QUIET can yield sufficient energy to support the growth of both syntrophic partners, but that the mechanisms by which electrons are derived from extracellular hydroquinones require further investigation.

Evidence for interaction between the triheme cytochrome PpcA from Geobacter sulfurreducens and anthrahydroquinone-2,6-disulfonate, an analog of the redox active components of humic substances

The bacterium Geobacter sulfurreducens displays an extraordinary respiratory versatility underpinning the diversity of electron donors and acceptors that can be used to sustain anaerobic growth. Remarkably, G. sulfurreducens can also use as electron donors the reduced forms of some acceptors, such as the humic substance analog anthraquinone-2,6-disulfonate (AQDS), a feature that confers environmentally competitive advantages to the organism. Using UV–visible and stopped-flow kinetic measurements we demonstrate that there is electron exchange between the triheme cytochrome PpcA from Gs and AQDS. 2D-1H–15N HSQC NMR spectra were recorded for 15N-enriched PpcA samples, in the absence and presence of AQDS. Chemical shift perturbation measurements, at increasing concentration of AQDS, were used to probe the interaction region and to measure the binding affinity of the PpcA–AQDS complex. The perturbations on the NMR signals corresponding to the PpcA backbone NH and heme substituents showed that the region around heme IV interacts with AQDS through the formation of a complex with a definite life time in the NMR time scale. The comparison of the NMR data obtained for PpcA in the presence and absence of AQDS showed that the interaction is reversible. Overall, this study provides for the first time a clear illustration of the formation of an electron transfer complex between AQDS and a G. sulfurreducens triheme cytochrome, shedding light on the electron transfer pathways underlying the microbial oxidation of humics.

Smectite Reduction by Shewanella Species as Facilitated by Cystine and Cysteine

Exogenous electron transfer mediators employed by Fe(III)-reducing bacteria are believed to govern the kinetics and equilibrium of bioreduction of Fe(III) in solid phase. In contrast to a large number of studies on humic substances and analog anthraquinone-2,6-disulfonate (AQDS), our knowledge of other potential electron shuttles involved in Fe(III) reduction is limited. The purpose of the present study was to understand the role of cystine and cysteine in reduction of iron-rich smectite (nontronite, NAu-2) by Shewanella species. A series of abiotic and biotic experiments were conducted in nongrowth media (bicarbonate buffered, pH = 7.0). Fe(II) and cysteine concentrations were monitored over the course of the bioreduction experiments with wet chemistry, and the unreduced and reduced nontronites were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results indicated that all Shewanella species tested here were capable of reducing cystine to cysteine. Either cystine or cysteine amendments significantly stimulated the Fe(III) bioreduction rate and extent. The initial reduction rate was linearly correlated with cystine or cysteine concentration. The reduction extent (18.7–22.3%) calculated from bioreactor with cystine or cysteine was slightly lower than those with AQDS (26.3%). Mineralogical analysis demonstrated that cystine or cysteine facilitated the reaction of smectite to illite as a result of Fe(III) bioreduction. Thus, we concluded that, in our experiments, cystine and cysteine functioned as electron carrier in the smectite reduction systems, and were favorable factors influencing smectite illitization.

Humic analog AQDS and AQS as an electron mediator can enhance chromate reduction by Bacillus sp. strain 3C3

Humus as an electron mediator is recognized as an effective strategy to improve the biological transformation and degradation of toxic substances, yet the action of humus in microbial detoxification of chromate is still unknown. In this study, a humus-reducing strain 3C(3) was isolated from mangrove sediment. Based on the analyses of morphology, physiobiochemical characteristics, and 16S rRNA gene sequence, this strain was identified Bacillus sp. Strain 3C(3) can effectively reduce humic analog anthraquinone-2,6-disulfonate (AQDS) and anthraquinone-2-sulfonate (AQS) with lactate, formate, or glucose as electron donors. When the cells were killed by incubation at 95°C for 30 min or an electron donor was absent, the humic reduction did not occur, showing that the humic reduction was a biochemical process. However, strain 3C(3) had low capability of chromate reduction under anaerobic conditions, despite of having strong tolerance of the toxic metal. But in the presence of humic substances AQDS or AQS, we found that chromate reduction by strain 3C(3) was enhanced greatly. Because strain 3C(3) is an effective humus-reducing bacterium, it is proposed that humic substances could serve as electron mediator to interact with chromate and accelerate chromate reduction. Our results suggest that chromate contaminations can be detoxified by adding humic analog (low to 0.1 mM) as an electron mediator in the microbial incubation.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) Biodegradation Kinetics Amongst Several Fe(III)-Reducing Genera

Cyclic nitramine explosives biodegradation was investigated among four Fe(III)- and quinone-reducing bacterial genera. This strategy is an attractive option for RDX and/or HMX contamination because of their ubiquity; however, the biotransformation kinetics among different microbial populations is not known. The organisms investigated included two species within the Geobacteraceae, and one species each within the genera Anaeromyxobacter, Desulfitobacterium, and Shewanella. All species directly reduced RDX; however, humic substances (HS) and the HS analog anthraquinone-2,6-disulfonate (AQDS) significantly increased the rate and extent of RDX reduction. Degradation kinetics varied amongst the species tested, but extracellular electron shuttle mediated degradation rates were the fastest for each organism. RDX reduction rates ranged from 7.4 to 269.3 nmol RDX hr − 1 mg cell protein − 1 when AQDS was present. HMX was reduced more slowly by G. metallireducens than RDX; however, electron shuttles also stimulated HMX degradation. These data suggest that electron shuttle mediated cyclic nitramine transformation is ubiquitous among the keystone Fe(III)-reducing microbial genera, and that bioremediation strategies predicated on their physiology may be a reasonable approach in situ for both Fe(III)-rich and Fe(III)-poor environments.

Sustained generation of electricity by the spore-forming, Gram-positive, Desulfitobacterium hafniense strain DCB2

Desulfitobacterium hafniense strain DCB2 generates electricity in microbial fuel cells (MFCs) when humic acids or the humate analog anthraquinone-2,6-disulfonate (AQDS) is added as an electron-carrying mediator. When utilizing formate as fuel, the Gram-positive, spore-forming bacterium generated up to 400 mW/m2 of cathode surface area in a single-chamber MFC with a platinum-containing air-fed cathode. Hydrogen, lactate, pyruvate, and ethanol supported electricity generation, but acetate, propionate, and butyrate did not. Scanning electron microscopy indicated that strain DCB2 colonized the surface of a current-generating anode but not of an unconnected electrode. The electricity was recovered fully within minutes after the exchange of the medium in the anode chamber and within a week after an exposure of a colonized anode to 90 degrees C for 20 min. Of the six strains of Desulfitobacteria tested, all of which would reduce AQDS, only D. hafniense strain DCB2 continued to reduce AQDS and generate electricity for more than 24 h, indicating that reduction of the humate analog alone is insufficient to sustain electrode reduction.

Shewanella oneidensis MR-1 restores menaquinone synthesis to a menaquinone-negative mutant.

The mechanisms underlying the use of insoluble electron acceptors by metal-reducing bacteria, such as Shewanella oneidensis MR-1, are currently under intensive study. Current models for shuttling electrons across the outer membrane (OM) of MR-1 include roles for OM cytochromes and the possible excretion of a redox shuttle. While MR-1 is able to release a substance that restores the ability of a menaquinone (MK)-negative mutant, CMA-1, to reduce the humic acid analog anthraquinone-2,6-disulfonate (AQDS), cross-feeding experiments conducted here showed that the substance released by MR-1 restores the growth of CMA-1 on several soluble electron acceptors. Various strains derived from MR-1 also release this substance; these include mutants lacking the OM cytochromes OmcA and OmcB and the OM protein MtrB. Even though strains lacking OmcB and MtrB cannot reduce Fe(III) or AQDS, they still release a substance that restores the ability of CMA-1 to use MK-dependent electron acceptors, including AQDS and Fe(III). Quinone analysis showed that this released substance restores MK synthesis in CMA-1. This ability to restore MK synthesis in CMA-1 explains the cross-feeding results and challenges the previous hypothesis that this substance represents a redox shuttle that facilitates metal respiration.

Electron transfer by Desulfobulbus propionicus to Fe(III) and graphite electrodes.

Desulfobulbus propionicus was able to grow with Fe(III), the humic acids analog anthraquinone-2,6-disulfonate (AQDS), or a graphite electrode as an electron acceptor. These results provide an explanation for the enrichment of Desulfobulbaceae species on the surface of electrodes harvesting electricity from anaerobic marine sediments and further expand the diversity of microorganisms known to have the ability to use both sulfate and Fe(III) as an electron acceptor.

Characterization of Fe(III) reduction by chlororespiring Anaeromyxobacter dehalogenans.

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [(14)C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.

Protective Role of tolC in Efflux of the Electron Shuttle Anthraquinone-2,6-Disulfonate

Extracellular electron transfer can play an important role in microbial respiration on insoluble minerals. The humic acid analog anthraquinone-2,6-disulfonate (AQDS) is commonly used as an electron shuttle during studies of extracellular electron transfer. Here we provide genetic evidence that AQDS enters Shewanella oneidensis strain MR-1 and causes cell death if it accumulates past a critical concentration. A tolC homolog protects the cell from toxicity by mediating the efflux of AQDS. Electron transfer to AQDS appears to be independent of the tolC pathway, however, and requires the outer membrane protein encoded by mtrB. We suggest that there may be structural and functional relationships between quinone-containing electron shuttles and antibiotics.

Reduction of Fe(III), Cr(VI), U(VI), and Tc(VII) by Deinococcus radiodurans R1.

Deinococcus radiodurans is an exceptionally radiation-resistant microorganism capable of surviving acute exposures to ionizing radiation doses of 15,000 Gy and previously described as having a strictly aerobic respiratory metabolism. Under strict anaerobic conditions, D. radiodurans R1 reduced Fe(III)-nitrilotriacetic acid coupled to the oxidation of lactate to CO(2) and acetate but was unable to link this process to growth. D. radiodurans reduced the humic acid analog anthraquinone-2,6-disulfonate (AQDS) to its dihydroquinone form, AH(2)DS, which subsequently transferred electrons to the Fe(III) oxides hydrous ferric oxide and goethite via a previously described electron shuttle mechanism. D. radiodurans reduced the solid-phase Fe(III) oxides in the presence of either 0.1 mM AQDS or leonardite humic acids (2 mg ml(-1)) but not in their absence. D. radiodurans also reduced U(VI) and Tc(VII) in the presence of AQDS. In contrast, Cr(VI) was directly reduced in anaerobic cultures with lactate although the rate of reduction was higher in the presence of AQDS. The results are the first evidence that D. radiodurans can reduce Fe(III) coupled to the oxidation of lactate or other organic compounds. Also, D. radiodurans, in combination with humic acids or synthetic electron shuttle agents, can reduce U and Tc and thus has potential applications for remediation of metal- and radionuclide-contaminated sites where ionizing radiation or other DNA-damaging agents may restrict the activity of more sensitive organisms.