Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture

A system for growing Geobacter sulfurreducens under anaerobic conditions in chemostats was developed in order to study the physiology of this organism under conditions that might more closely approximate those found in the subsurface than batch cultures. Geobacter sulfurreducens could be cultured under acetate-limiting conditions with fumarate or Fe(III)-citrate as the electron acceptor at growth rates between 0.04 and 0.09 h(-1). The molar growth yield was threefold higher with fumarate as the electron acceptor than with Fe(III), despite the lower mid-point potential of the fumarate/succinate redox couple. When growth was limited by availability of fumarate, high steady-state concentrations were detected, suggesting that fumarate is unlikely to be an important electron acceptor in sedimentary environments. The half-saturation constant, Ks, for acetate in Fe(III)-grown cultures (10 microM) suggested that the growth of Geobacter species is likely to be acetate limited in most subsurface sediments, but that when millimolar quantities of acetate are added to the subsurface in order to promote the growth of Geobacter for bioremediation applications, this should be enough to overcome any acetate limitations. When the availability of electron acceptors, rather than acetate, limited growth, G. sulfurreducens was less efficient in incorporating acetate into biomass but had higher respiration rates, a desirable physiological characteristic when adding acetate to stimulate the activity of Geobacter species during in situ uranium bioremediation. These results demonstrate that the ability to study the growth of G. sulfurreducens under steady-state conditions can provide insights into its physiological characteristics that have relevance for its activity in a diversity of sedimentary environments.     

Genetic characterization of a single bifunctional enzyme for fumarate reduction and succinate oxidation in Geobacter sulfurreducens and engineering of fumarate reduction in Geobacter metallireducens

The mechanism of fumarate reduction in Geobacter sulfurreducens was investigated. The genome contained genes encoding a heterotrimeric fumarate reductase, FrdCAB, with homology to the fumarate reductase of Wolinella succinogenes and the succinate dehydrogenase of Bacillus subtilis. Mutation of the putative catalytic subunit of the enzyme resulted in a strain that lacked fumarate reductase activity and was unable to grow with fumarate as the terminal electron acceptor. The mutant strain also lacked succinate dehydrogenase activity and did not grow with acetate as the electron donor and Fe(III) as the electron acceptor. The mutant strain could grow with acetate as the electron donor and Fe(III) as the electron acceptor if fumarate was provided to alleviate the need for succinate dehydrogenase activity in the tricarboxylic acid cycle. The growth rate of the mutant strain under these conditions was faster and the cell yields were higher than for wild type grown under conditions requiring succinate dehydrogenase activity, suggesting that the succinate dehydrogenase reaction consumes energy. An orthologous frdCAB operon was present in Geobacter metallireducens, which cannot grow with fumarate as the terminal electron acceptor. When a putative dicarboxylic acid transporter from G. sulfurreducens was expressed in G. metallireducens, growth with fumarate as the sole electron acceptor was possible. These results demonstrate that, unlike previously described organisms, G. sulfurreducens and possibly G. metallireducens use the same enzyme for both fumarate reduction and succinate oxidation in vivo.     

Electrochemical characterization of Geobacter sulfurreducens cells immobilized on graphite paper electrodes

Bacteria able to transfer electrons to conductive surfaces are of interest as catalysts in microbial fuel cells, as well as in bioprocessing, bioremediation, and corrosion. New procedures for immobilization of Geobacter sulfurreducens on graphite electrodes are described that allow routine, repeatable electrochemical analysis of cell-electrode interactions. Immediately after immobilizing G. sulfurreducens on electrodes, electrical current was obtained without addition of exogenous electron shuttles or electroactive polymers. Voltammetry and impedance analysis of pectin-immobilized bacteria transferring electrons to electrode surfaces could also be performed. Cyclic voltammetry of immobilized cells revealed voltage-dependent catalytic current similar to what is commonly observed with adsorbed enzymes, with catalytic waves centered at -0.15 V (vs. SHE). Electrodes maintained at +0.25 V (vs. SHE) initially produced 0.52 A/m(2) in the presence of acetate as the electron donor. Electrical Impedance Spectroscopy of coatings was also consistent with a catalytic mechanism, controlled by charge transfer rate. When electrodes were maintained at an oxidizing potential for 24 h, electron transfer to electrodes increased to 1.75 A/m(2). These observations of electron transfer by pectin-entrapped G. sulfurreducens appear to reflect native mechanisms used for respiration. The ability of washed G. sulfurreducens cells to immediately produce electrical current was consistent with the external surface of this bacterium possessing a pathway linking oxidative metabolism to extracellular electron transfer. This electrochemical activity of pectin-immobilized bacteria illustrates a strategy for preparation of catalytic electrodes and study of Geobacter under defined conditions.     

Graphite electrode as a sole electron donor for reductive dechlorination of tetrachlorethene by Geobacter lovleyi

The possibility that graphite electrodes can serve as the direct electron donor for microbially catalyzed reductive dechlorination was investigated with Geobacter lovleyi. In an initial evaluation of whether G. lovleyi could interact electronically with graphite electrodes, cells were provided with acetate as the electron donor and an electrode as the sole electron acceptor. Current was produced at levels that were ca. 10-fold lower than those previously reported for Geobacter sulfurreducens under similar conditions, and G. lovleyi anode biofilms were correspondingly thinner. When an electrode poised at -300 mV (versus a standard hydrogen electrode) was provided as the electron donor, G. lovleyi effectively reduced fumarate to succinate. The stoichiometry of electrons consumed to succinate produced was 2:1, the ratio expected if the electrode served as the sole electron donor for fumarate reduction. G. lovleyi effectively reduced tetrachloroethene (PCE) to cis-dichloroethene with a poised electrode as the sole electron donor at rates comparable to those obtained when acetate serves as the electron donor. Cells were less abundant on the electrodes when the electrodes served as an electron donor than when they served as an electron acceptor. PCE was not reduced in controls without cells or when the current supply to cells was interrupted. These results demonstrate that G. lovleyi can use a poised electrode as a direct electron donor for reductive dechlorination of PCE. The ability to colocalize dechlorinating microorganisms with electrodes has several potential advantages for bioremediation of subsurface chlorinated contaminants, especially in source zones where electron donor delivery is challenging and often limits dechlorination.     

Elucidation of an alternate isoleucine biosynthesis pathway in Geobacter sulfurreducens

The central metabolic model for Geobacter sulfurreducens included a single pathway for the biosynthesis of isoleucine that was analogous to that of Escherichia coli, in which the isoleucine precursor 2-oxobutanoate is generated from threonine. 13C labeling studies performed in G. sulfurreducens indicated that this pathway accounted for a minor fraction of isoleucine biosynthesis and that the majority of isoleucine was instead derived from acetyl-coenzyme A and pyruvate, possibly via the citramalate pathway. Genes encoding citramalate synthase (GSU1798), which catalyzes the first dedicated step in the citramalate pathway, and threonine ammonia-lyase (GSU0486), which catalyzes the conversion of threonine to 2-oxobutanoate, were identified and knocked out. Mutants lacking both of these enzymes were auxotrophs for isoleucine, whereas single mutants were capable of growth in the absence of isoleucine. Biochemical characterization of the single mutants revealed deficiencies in citramalate synthase and threonine ammonia-lyase activity. Thus, in G. sulfurreducens, 2-oxobutanoate can be synthesized either from citramalate or threonine, with the former being the main pathway for isoleucine biosynthesis. The citramalate synthase of G. sulfurreducens constitutes the first characterized member of a phylogenetically distinct clade of citramalate synthases, which contains representatives from a wide variety of microorganisms.     

Identification of an extracellular polysaccharide network essential for cytochrome anchoring and biofilm formation in Geobacter sulfurreducens

Transposon insertions in Geobacter sulfurreducens GSU1501, part of an ATP-dependent exporter within an operon of polysaccharide biosynthesis genes, were previously shown to eliminate insoluble Fe(III) reduction and use of an electrode as an electron acceptor. Replacement of GSU1501 with a kanamycin resistance cassette produced a similarly defective mutant, which could be partially complemented by expression of GSU1500 to GSU1505 in trans. The Δ1501 mutant demonstrated limited cell-cell agglutination, enhanced attachment to negatively charged surfaces, and poor attachment to positively charged poly-d-lysine- or Fe(III)-coated surfaces. Wild-type and mutant cells attached to graphite electrodes, but when electrodes were poised at an oxidizing potential inducing a positive surface charge (+0.24 V versus the standard hydrogen electrode [SHE]), Δ1501 mutant cells detached. Scanning electron microscopy revealed fibrils surrounding wild-type G. sulfurreducens which were absent from the Δ1501 mutant. Similar amounts of type IV pili and pilus-associated cytochromes were detected on both cell types, but shearing released a stable matrix of c-type cytochromes and other proteins bound to polysaccharides. The matrix from the mutant contained 60% less sugar and was nearly devoid of c-type cytochromes such as OmcZ. The addition of wild-type extracellular matrix to Δ1501 cultures restored agglutination and Fe(III) reduction. The polysaccharide binding dye Congo red preferentially bound wild-type cells and extracellular matrix material over mutant cells, and Congo red inhibited agglutination and Fe(III) reduction by wild-type cells. These results demonstrate a crucial role for the xap (extracellular anchoring polysaccharide) locus in metal oxide attachment, cell-cell agglutination, and localization of essential cytochromes beyond the Geobacter outer membrane.     

Microbial fuel cell biosensor for in situ assessment of microbial activity

Microbial fuel cell (MFC)-based sensing was explored to provide useful information for the development of an approach to in situ monitoring of substrate concentration and microbial respiration rate. The ability of a MFC to provide meaningful information about in situ microbial respiration and analyte concentration was examined in column systems, where Geobacter sulfurreducens used an external electron acceptor (an electrode) to metabolize acetate. Column systems inoculated with G. sulfurreducens were operated with influent media at varying concentrations of acetate and monitored for current generation. Current generation was mirrored by bulk phase acetate concentration, and a correlation (R(2)=0.92) was developed between current values (0-0.30 mA) and acetate concentrations (0-2.3 mM). The MFC-system was also exposed to shock loading (pulses of oxygen), after which electricity production resumed immediately after media flow recommenced, underlining the resilience of the system and allowing for additional sensing capacity. Thus, the electrical signal produced by the MFC-system provided real-time data for electron donor availability and biological activity. These results have practical implications for development of a biosensor for inexpensive real-time monitoring of in situ bioremediation processes, where MFC technology provides information on the rate and nature of biodegradation processes.     

Lack of electricity production by Pelobacter carbinolicus indicates that the capacity for Fe(III) oxide reduction does not necessarily confer electron transfer ability to fuel cell anodes

The ability of Pelobacter carbinolicus to oxidize electron donors with electron transfer to the anodes of microbial fuel cells was evaluated because microorganisms closely related to Pelobacter species are generally abundant on the anodes of microbial fuel cells harvesting electricity from aquatic sediments. P. carbinolicus could not produce current in a microbial fuel cell with electron donors which support Fe(III) oxide reduction by this organism. Current was produced using a coculture of P. carbinolicus and Geobacter sulfurreducens with ethanol as the fuel. Ethanol consumption was associated with the transitory accumulation of acetate and hydrogen. G. sulfurreducens alone could not metabolize ethanol, suggesting that P. carbinolicus grew in the fuel cell by converting ethanol to hydrogen and acetate, which G. sulfurreducens oxidized with electron transfer to the anode. Up to 83% of the electrons available in ethanol were recovered as electricity and in the metabolic intermediate acetate. Hydrogen consumption by G. sulfurreducens was important for ethanol metabolism by P. carbinolicus. Confocal microscopy and analysis of 16S rRNA genes revealed that half of the cells growing on the anode surface were P. carbinolicus, but there was a nearly equal number of planktonic cells of P. carbinolicus. In contrast, G. sulfurreducens was primarily attached to the anode. P. carbinolicus represents the first Fe(III) oxide-reducing microorganism found to be unable to produce current in a microbial fuel cell, providing the first suggestion that the mechanisms for extracellular electron transfer to Fe(III) oxides and fuel cell anodes may be different.     

Characterization of metabolism in the Fe(III)-reducing organism Geobacter sulfurreducens by constraint-based modeling

Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.     

Fluorescent properties of c-type cytochromes reveal their potential role as an extracytoplasmic electron sink in Geobacter sulfurreducens

A novel fluorescence technique for monitoring the redox status of c-type cytochromes in Geobacter sulfurreducens was developed in order to evaluate the capacity of these extracytoplasmic cytochromes to store electrons during periods in which an external electron acceptor is not available. When intact cells in which the cytochromes were in a reduced state were excited at a wavelength of 350 nm, they fluoresced with maxima at 402 and 437 nm. Oxidation of the cytochromes resulted in a loss of fluorescence. This method was much more sensitive than the traditional approach of detecting c-type cytochromes via visible light absorbance. Furthermore, fluorescence of reduced cytochromes in individual cells could be detected via fluorescence microscopy, and the cytochromes in a G. sulfurreducens biofilm, remotely excited with an optical fibre, could be detected at distances as far as 5 cm. Fluorescence analysis of cytochrome oxidation and reduction of the external electron acceptor, anthraquinone-2,6-disulfonate, suggested that the extracytoplasmic cytochromes of G. sulfurreducens could store approximately 10(7) electrons per cell. Independent analysis of the haem content of the cells determined from analysis of incorporation of (55)Fe into cytochromes provided a similar estimate of cytochrome electron-storage capacity. This electron-storage capacity could, in the absence of an external electron acceptor, permit continued electron transfer across the inner membrane sufficient to supply the maintenance energy requirements for G. sulfurreducens for up to 8 min or enough proton motive force to power flagella motors for G. sulfurreducens motility. The fluorescence approach described here provides a sensitive method for evaluating the redox status of Geobacter species in culture and/or its environments. Furthermore, these results suggest that the periplasmic and outer-membrane cytochromes of Geobacter species act as capacitors, allowing continued electron transport, and thus viability and motility, for Geobacter species as they move between heterogeneously dispersed Fe(III) oxides during growth in the subsurface.     

OmcB,a c-type polyheme cytochrome,involved in Fe(III) reduction in Geobacter sulfurreducens

Microorganisms in the family Geobacteraceae are the predominant Fe(III)-reducing microorganisms in a variety of subsurface environments in which Fe(III) reduction is an important process, but little is known about the mechanisms for electron transport to Fe(III) in these organisms. The Geobacter sulfurreducens genome was found to contain a 10-kb chromosomal duplication consisting of two tandem three-gene clusters. The last genes of the two clusters, designated omcB and omcC, encode putative outer membrane polyheme c-type cytochromes which are 79% identical. The role of the omcB and omcC genes in Fe(III) reduction in G. sulfurreducens was investigated. OmcB and OmcC were both expressed during growth with acetate as the electron donor and either fumarate or Fe(III) as the electron acceptor. OmcB was ca. twofold more abundant under both conditions. Disrupting omcB or omcC by gene replacement had no impact on growth with fumarate. However, the OmcB-deficient mutant was greatly impaired in its ability to reduce Fe(III) both in cell suspensions and under growth conditions. In contrast, the ability of the OmcC-deficient mutant to reduce Fe(III) was similar to that of the wild type. When omcB was reintroduced into the OmcB-deficient mutant, the capacity for Fe(III) reduction was restored in proportion to the level of OmcB production. These results indicate that OmcB, but not OmcC, has a major role in electron transport to Fe(III) and suggest that electron transport to the outer membrane is an important feature in Fe(III) reduction in this organism.     

Electricity production by Geobacter sulfurreducens attached to electrodes

Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 micro M), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 micro mol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (E(o)' =+0.37 V). The production of current in microbial fuel cell (65 mA/m(2) of electrode surface) or poised-potential (163 to 1,143 mA/m(2)) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G. sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.     

Proteome of Geobacter sulfurreducens grown with Fe(III) oxide or Fe(III) citrate as the electron acceptor

The mechanisms for Fe(III) oxide reduction in Geobacter species are of interest because Fe(III) oxides are the most abundant form of Fe(III) in many soils and sediments and Geobacter species are prevalent Fe(III)-reducing microorganisms in many of these environments. Protein abundance in G. sulfurreducens grown on poorly crystalline Fe(III) oxide or on soluble Fe(III) citrate was compared with a global accurate mass and time tag proteomic approach in order to identify proteins that might be specifically associated with Fe(III) oxide reduction. A total of 2991 proteins were detected in G. sulfurreducens grown with acetate as the electron donor and either Fe(III) oxide or soluble Fe(III) citrate as the electron acceptor, resulting in 86% recovery of the genes predicted to encode proteins. Of the total expressed proteins 76% were less abundant in Fe(III) oxide cultures than in Fe(III) citrate cultures, which is consistent with the overall slower rate of metabolism during growth with an insoluble electron acceptor. A total of 269 proteins were more abundant in Fe(III) oxide-grown cells than in cells grown on Fe(III) citrate. Most of these proteins were in the energy metabolism category: primarily electron transport proteins, including 13 c-type cytochromes and PilA, the structural protein for electrically conductive pili. Several of the cytochromes that were more abundant in Fe(III) oxide-grown cells were previously shown with genetic approaches to be essential for optimal Fe(III) oxide reduction. Other proteins that were more abundant during growth on Fe(III) oxide included transport and binding proteins, proteins involved in regulation and signal transduction, cell envelope proteins, and enzymes for amino acid and protein biosynthesis, among others. There were also a substantial number of proteins of unknown function that were more abundant during growth on Fe(III) oxide. These results indicate that electron transport to Fe(III) oxide requires additional and/or different proteins than electron transfer to soluble, chelated Fe(III) and suggest proteins whose functions should be further investigated in order to better understand the mechanisms of electron transfer to Fe(III) oxide in G. sulfurreducens.     

Geobacter sulfurreducens sp. nov., a hydrogen- and acetate-oxidizing dissimilatory metal-reducing microorganism.

A dissimilatory metal- and sulfur-reducing microorganism was isolated from surface sediments of a hydrocarbon-contaminated ditch in Norman, Okla. The isolate, which was designated strain PCA, was an obligately anaerobic, nonfermentative nonmotile, gram-negative rod. PCA grew in a defined medium with acetate as an electron donor and ferric PPi, ferric oxyhydroxide, ferric citrate, elemental sulfur, Co(III)-EDTA, fumarate, or malate as the sole electron acceptor. PCA also coupled the oxidation of hydrogen to the reduction of Fe(III) but did not reduce Fe(III) with sulfur, glucose, lactate, fumarate, propionate, butyrate, isobutyrate, isovalerate, succinate, yeast extract, phenol, benzoate, ethanol, propanol, or butanol as an electron donor. PCA did not reduce oxygen, Mn(IV), U(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PCA exhibited dithionite-reduced minus air-oxidized difference spectra which were characteristic of c-type cytochromes. Phylogenetic analysis of the 16S rRNA sequence placed PCA in the delta subgroup of the proteobacteria. Its closest known relative is Geobacter metallireducens. The ability to utilize either hydrogen or acetate as the sole electron donor for Fe(III) reduction makes strain PCA a unique addition to the relatively small group of respiratory metal-reducing microorganisms available in pure culture. A new species name, Geobacter sulfurreducens, is proposed.     

The RpoS sigma factor in the dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens

Geobacter sulfurreducens RpoS sigma factor was shown to contribute to survival in stationary phase and upon oxygen exposure. Furthermore, a mutation in rpoS decreased the rate of reduction of insoluble Fe(III) but not of soluble forms of iron. This study suggests that RpoS plays a role in regulating metabolism of Geobacter under suboptimal conditions in subsurface environments.     

Lactate oxidation coupled to iron or electrode reduction by Geobacter sulfurreducens PCA

Geobacter sulfurreducens PCA completely oxidized lactate and reduced iron or an electrode, producing pyruvate and acetate intermediates. Compared to the current produced by Shewanella oneidensis MR-1, G. sulfurreducens PCA produced 10-times-higher current levels in lactate-fed microbial electrolysis cells. The kinetic and comparative analyses reported here suggest a prominent role of G. sulfurreducens strains in metal- and electrode-reducing communities supplied with lactate.     

Cysteine-mediated electron transfer in syntrophic acetate oxidation by cocultures of Geobacter sulfurreducens and Wolinella succinogenes

Syntrophic cocultures of Geobacter sulfurreducens and Wolinella succinogenes oxidize acetate with nitrate as terminal electron acceptor. It has been postulated earlier that electrons are transferred in these cocultures not via hydrogen, but via a different carrier, e.g., a small c-type cytochrome that is detected in the supernatant of growing cultures. In the present study, L -cysteine, which was provided as a reducing agent, was found to mediate the electron transfer between the two partners. Low concentrations of L -cysteine or L -cystine (10-100 microM) supported syntrophic growth, and no acetate oxidation was observed in the absence of cysteine or cystine. Cell suspensions of G. sulfurreducens or coculture cell suspensions reduced cystine to cysteine, and suspensions of W. succinogenes or coculture suspensions oxidized cysteine with nitrate, as measured by the formation or depletion of free thiol groups. Added cysteine was rapidly oxidized by the coculture during growth, but the formed cystine was not entirely rereduced even under acceptor-limited conditions. The redox potential prevailing in acetate-oxidizing cocultures was -160 to -230 mV. Sulfide at low concentrations supported syntrophic growth as well and could replace cysteine. Neither growth nor acetate degradation was found with D-cysteine, homocysteine, cysteamine, 3-mercaptopropionate, dithiothreithol, thioglycolate, glutathione, coenzyme M, dimethylsulfoxide, trimethylamine- N-oxide, anthraquinone-2,6-disulfonate, or ascorbate.