Cooperation between autotrophic and heterotrophic denitrifiers under low C/N ratios revealed by individual-based modelling.

Denitrifying biofilms, in which autotrophic denitrifiers (AD) and heterotrophic denitrifiers (HD) coexist, play a crucial role in removing nitrate from water or wastewater. However, it is difficult to elucidate the interactions between HD and AD through sequencing-based experimental methods. Here, we developed an individual-based model to describe the interspecies dynamics and priority effects between sulfur-based AD (Thiobacillus denitrificans) and HD (Thauera phenylcarboxya) under different C/N ratios. In test I (coexistence simulation), AD and HD were initially inoculated at a ratio of 1:1. The simulation results showed excellent denitrification performance and a coaggregation pattern of denitrifiers, indicating that cooperation was the predominant interaction at a C/N ratio of 0.25 to 1.5. In test II (invasion simulation), in which only one type of denitrifier was initially inoculated and the other was added at the invasion time, denitrifiers exhibited a stratification pattern in biofilms. When HD invaded AD, the final HD abundance decreased with increasing invasion time, indicating an enhanced priority effect. When AD invaded HD, insufficient organic carbon sources weakened the priority effect by limiting the growth of HD populations. This study reveals the interaction between autotrophic and heterotrophic denitrifiers, providing guidance for optimizing wastewater treatment process.

U mobilization and associated U isotope fractionation by sulfur-oxidizing bacteria.

Uranium (U) contamination of the environment causes high risk to health, demanding for effective and sustainable remediation. Bioremediation via microbial reduction of soluble U(VI) is generating high fractions (>50%) of insoluble non-crystalline U(IV) which, however, might be remobilized by sulfur-oxidizing bacteria. In this study, the efficacy of Acidithiobacillus (At.) ferrooxidans and Thiobacillus (T.) denitrificans to mobilize non-crystalline U(IV) and associated U isotope fractionation were investigated. At. ferrooxidans mobilized between 74 and 91% U after 1 week, and U mobilization was observed for both, living and inactive cells. Contrary to previous observations, no mobilization by T. denitrificans could be observed. Uranium mobilization by At. ferrooxidans did not cause U isotope fractionation suggesting that U isotope ratio determination is unsuitable as a direct proxy for bacterial U remobilization. The similar mobilization capability of active and inactive At. ferrooxidans cells suggests that the mobilization is based on the reaction with the cell biomass. This study raises doubts about the long-term sustainability of in-situ bioremediation measures at U-contaminated sites, especially with regard to non-crystalline U(IV) being the main component of U bioremediation.

Synergistic between autotrophic and heterotrophic microorganisms for denitrification using bio-S as electron donor.

In recent years, biological sulfur (bio-S) was employed in sulfur autotrophic denitrification (SAD) in which autotrophic Thiobacillus denitrificans and heterotrophic Stenotrophomonas maltophilia played a key role. The growth pattern of T.denitrificans and S.maltophilia exhibited a linear relationship between OD600 and CFU when OD600 < 0.06 and <0.1, respectively. When S.maltophilia has applied alone, the NorBC and NosZ were undetected, and denitrification was incomplete. The DsrA of S.maltophilia could produce sulfide as an alternative electron donor for T.denitrificans. Even though T.denitrificans had complete denitrification genes, its efficiency was low when used alone. The interaction of T.denitrificans and S.maltophilia reduced nitrite accumulation, leading to complete denitrification. A sufficient quantity of S.maltophilia may trigger the autotrophic denitrification activity of T.denitrificans. When the colony-forming units (CFU) ratio of S.maltophilia to T.denitrificans was reached at 2:1, the highest denitrification performance was achieved at 2.56 and 12.59 times higher than applied alone. This research provides a good understanding of the optimal microbial matching for the future application of bio-S.

Nanoplastics impacts on Thiobacillus denitrificans: Effects of size and dissolved organic matter.

The widespread distribution of nanoplastics and dissolved organic matter (DOM) in sewage raises concerns about the potential impact of DOM on the bioavailability of nanoplastics. In this study, the effects of different sizes (100 nm and 350 nm) of polystyrene nanoplastics (PS-NPs, 50 mg/L) and combined with 10 mg/L or 50 mg/L DOMs (fulvic acid, humic acid and sodium alginate) on the growth and denitrification ability of Thiobacillus denitrificans were investigated. Results showed that 100 nm PS-NPs (50 mg/L) cause a longer delay in the nitrate reduction (3 days) of T. denitrificans than 350 nm PS-NPs (2 days). Furthermore, the presence of DOM exacerbated the adverse effect of 100 nm PS-NPs on denitrification, resulting in a delay of 1-4 days to complete denitrification. Fulvic acid (50 mg/L) and humic acid (50 mg/L) had the most significant adverse effect on increasing 100 nm PS-NPs (50 mg/L), causing a reduction of 20 mmol/L nitrate by T. denitrificans in nearly 7 days. It is noteworthy that the presence of DOM did not modify the adverse effect of 350 nm PS-NPs on denitrification. Further analysis of toxicity mechanism of PS-NPs revealed that they could induce reactive oxygen species (ROS) and suppressed denitrification gene expression. The results suggested that DOM may assist in the cellular internalization of PS-NPs by inhibiting PS-NPs aggregation, leading to the increased ROS levels and accelerated T. denitrificans death. This study highlights the potential risk of nanoplastics to autotrophic denitrifying bacteria in the presence of DOM and provides new insights for the treatment of nitrogen-containing wastewater by T. denitrificans.

Enhanced biological S0 accumulation by using signal molecules during simultaneous desulfurization and denitrification.

A high rate of elemental sulfur (S0) accumulation from sulfide-containing wastewater has great significance in terms of resource recovery and pollution control. This experimental study used Thiobacillus denitrificans and denitrifying bacteria incorporated with signal molecules (C6 and OHHL) for simultaneous sulfide (S2-) and nitrate (NO3-) removal in synthetic wastewater. Also, the effects on S0 accumulation due to changes in organic matter composition and bacteria proportion through signal molecules were analyzed. The 99.0% of S2- removal and 99.3% of NO3- was achieved with 66% of S0 accumulation under the active S2- removal group. The S0 accumulation, S2- and NO3- removal mainly occurred in 0-48 h. The S0 accumulation in the active S2- removal group was 2.0-6.3 times higher than the inactive S2- removal groups. In addition, S0/SO42- ratio exhibited that S0 conversion almost linearly increased with reaction time under the active S2- removal group. The proportion of Thiobacillus denitrificans and H+ consumption showed a positive correlation with S0 accumulation. However, a very high or low ratio of H+/S0 is not suitable for S0 accumulation. The signal molecules greatly increased the concentration of protein-I and protein-II, which resulted in the high proportion of Thiobacillus denitrificans. Therefore, high S0 accumulation was achieved as Thiobacillus denitrificans regulated the H+ consumption and electron transfer rate and provided suppressed oxygen environment. This technology is cost-effective and commercially applicable for recovering S0 from wastewater.

Dissolved Organic Matter Acting as a Microbial Photosensitizer Drives Photoelectrotrophic Denitrification.

The biogeochemical fates of dissolved organic matter (DOM) show important environmental significance in aqueous ecosystems. However, the current understanding of the trophic relationship between DOM and microorganisms limits the ability of DOM to serve as a heterotrophic substrate or electron shuttle for microorganisms. In this work, we provide the first evidence of photoelectrophy, a new trophic linkage, that occurs between DOM and nonphototrophic microorganisms. Specifically, the photoelectrotrophic denitrification process was demonstrated in a Thiobacillus denitrificans-DOM coupled system, in which DOM acted as a microbial photosensitizer to drive the model denitrifier nitrate reduction. The reduction of nitrate followed a pseudo-first-order reaction with a kinetic constant of 0.06 ± 0.003 h-1, and the dominant nitrogenous product was nitrogen. The significant upregulated (p < 0.01) expression of denitrifying genes, including nar, nir, nor, and nos, supported that the conversion of nitrate to nitrogen was the microorganism-mediated process. Interestingly, the photoelectrophic process triggered by DOM photosensitization promotes humification of DOM itself, an almost opposite trend of pure DOM irradiation. The finding not only reveals a so far overlooked role of DOM serving as the microbial photosensitizer in sunlit aqueous ecosystems but also suggests a strategy for promoting sunlight-driven denitrification in surface environments.

Regulating intermediates to realize the coupled hydrion with biology polysulfide in wastewater treatment.

The purpose of this study is to clarify the mechanism of the coupled hydrion with biology polysulfide in the simultaneous denitrification and desulfurization process. The coupled hydrion with biology polysulfide, uncoupled hydrion with biology polysulfide and no polysulfide experiments were performed in wastewater with two kinds of sulfide loads (100 and 200 mg/L). When the concentration of thiosulfate was suitable, the free H+ concentration (74.2 and 91.0 mg/L) and the proportion of Thiobacillus denitrificans (85.4% and 59.7%) were both higher under the two kinds of sulfide loading conditions (100 and 200 mg/L), and coupled hydrion with biology polysulfide was realized (the production of elemental sulfur is as high as 33 and 101 mg/L). Further analysis shown that the way of coupled hydrion with biology polysulfide were both: 2.0S2-+6.4NO3-+30.1H++21.7e-→1.0S2-+1.0SO42-+3.2N2+15.0H2O. In addition, for the coupled hydrion with biology polysulfide, more nitrates could be utilized to produce elemental sulfur S0, and the lower ratio of H+/S0 and SO42-/S0 were observed (S2- = 100 mg/L: 2.3 and 0.9; S2- = 200 mg/L: 0.9 and 0.03), which could promote the growth of Thiobacillus denitrificans and increase the proportion of Thiobacillus denitrificans. This maybe one of the reasons why coupled hydrion with biology polysulfide could be achieved.

Effects of an organic carbon source on the coupling of sulfur(thiosulfate)-driven denitration with Anammox process.

The sulfur(thiosulfate)-driven autotrophic denitrification coupled with Anammox (SDDA) process is proposed as an emerging technology for wastewater containing NH4+-N and NO3--N. However, the influence of organic matter on the SDDA process is not fully understood. A long-term experiment has shown that a moderate organic (acetate) (＜140 mg/L COD) can accelerate the heterotrophic/autotrophic denitrification and Anammox activity, to reach as high as 92.8% ± 0.3% total nitrogen at a loading rate of 1.34 kg-N/(m3·d). Batch test results showed that Anammox made the largest contribution to the removal of nitrogen, even in an SDDA system with COD addition. Additionally, organics can promote the bioavailability of solid sulfur through reaction with sulfide to form polysulfides, which increased nitrite accumulation to forward Anammox process. Sulfur-oxidizing bacteria (e.g., Thiobacillus and Denitratisoma) coexisted with Anammox bacteria (e.g., Ca. Brocadia and Ca. Kuenenia) in the SDDA system despite the addition of exogenous COD.

Effects of coarse and fine bubble aeration on performances of membrane filtration and denitrification in moving bed membrane bioreactors.

In this study, two lab-scale Moving Bed Membrane Bioreactors (MBMBR) were setup and operated in parallel to study the effect of coarse and fine bubble aeration on the performances of membrane filtration and denitrification treating domestic wastewater. The bacterial populations in the two MBMBRs were further analyzed to investigate the mechanisms involved in the different denitrification performances. The results showed that coarse bubble aeration could effectively mitigate membrane fouling by decreasing the formation of cake layer, although smaller sizes of bio-flocs were induced. In addition, coarse bubble aeration could also maintain dissolved oxygen (DO) at a relatively lower level without compromising the moving of bio-carriers, which achieved 10% higher total nitrogen removal rate due to anoxic zone created at inner layers of biofilms on bio-carriers. Accumulation of denitrifier (Thiobacillus denitrificans) on the bio-carriers was found under the coarse bubble aeration system, which can explain its superior denitrification performance.

Microscopic analysis towards rhamnolipid-mediated adhesion of Thiobacillus denitrificans: A QCM-D study.

Rhamnolipid was proved to increase the abundance of Thiobacillus denitrificans in the mixotrophic denitrification biofilm while its microscopic mechanism remains to be explored. Effect of rhamnolipids on deposition of macromolecular substances and adhesion of Thiobacillus denitrificans at room (20 °C) and low temperature (10 °C) were systematically investigated by the quartz crystal microbalance with dissipation monitoring (QCM-D) for the first time. Results showed that low concentration of rhamnolipids (20-80 mg/L) could promote the deposition of macromolecular substances by reducing hydraulic repulsion force, with the maximum deposition amount increased by 4.28 times than that of the control at room temperature. Deposition amount of microorganisms could be improved by increasing its concentration at room temperature while it didn't work at low temperature. Meanwhile, low temperature could significantly inhibit adhesion of Thiobacillus denitrificans (p < 0.05) and deposited layers under low concentration of rhamnolipids were generally rigid, resulting in the negative feedback effect on the microorganisms' adhesion. While high concentration of rhamnolipids (120-200 mg/L) could regulate the biofilm from rigid to viscoelastic and significantly promote the initial adhesion of Thiobacillus denitrificans on SiO2 surface (p < 0.05). This study demonstrated the microscopic mechanism of rhamnolipids on the initial biofilm formation, that is, the reduction of hydration repulsion force was responsible for the enhanced deposition of macromolecules while the regulation of biofilm properties was account for the promoted adhesion of Thiobacillus denitrificans.

Organic carbon coupling with sulfur reducer boosts sulfur based denitrification by Thiobacillus denitrificans.

Sulfur autotrophic denitrification utilizes elemental sulfur as the electron donor for nitrate removal from aquatic environments. Organic carbon could stimulate the conversion of sulfur and facilitates the S0-based denitrification process in the mix-trophic. In this study, the co-cultured system of sulfur reducer (Geobacter sulfurreducens) and Thiobacillus denitrificans was used to investigate that how organic carbon could boost the S0-based denitrification. The results showed that the rate of S0-based denitrification was improved with C/N ratio of 0.13 and this improvement continued even after the acetate was exhausted. Sulfur probe test and Raman analysis suggested that reduced sulfur species (Sx2-) were formed with the addition of organic carbon. The Sx2- could recombine with element sulfur and the bioavailability of S0 would be improved, as a result, the rate of S0-based denitrification increased as well. Nitrate reduction rate could further increase with the C/N ratio of 0.88, but it would decrease significantly when the C/N ratio increased to 1.50 as the high concentration of generated S2-. Our results provided explanations that why organic carbon addition would improve the bioavailability of S0 which could further promote the S0-dominant denitrification process.

[Effect of Signal Molecule Combined with Thiobacillus denitrificans on Simultaneous Removal of Nitrogen and Sulfur].

The effects of Thiobacillus denitrificans combined with signal molecules on the removal of sulfide and nitrate was investigated. By adding signal molecules and T. denitrificans at the same, the total number of microorganisms increased, the removal of sulfide and nitrate was accelerated, and an increase in nitrogen gas and more stable accumulation of elemental sulfur was observed. The total number of microorganisms after the reaction was detected using fluorescence in situ hybridization (FISH) technique. In this experiment, the optimal concentration for the stable accumulation of elemental sulfur from six concentrations of signal molecules was revealed. Further, the effects of adding signal molecules, T. denitrificans, and their combination were analyzed at this concentration. The results showed that it was easier to accumulate elemental sulfur after the addition of 1.0 µmol·L-1 signal molecule. After adding both T. denitrificans and 1.0 µmol·L-1 signal molecules at a sulfide concentration of 200 mg·L-1, the removal of sulfide and nitrate increased to 99.8% and 96.9% at 72 h, respectively, and increases in nitrogen gas and sulfur were observed. The amounts of elemental sulfur and nitrogen gas reached to 59.0 mg and 80.0 mL, respectively, after adding 2.5 µmol·L-1 signal molecules at 72 h when the sulfide concentration was 300 mg·L-1. Under those conditions, the removal efficiency of sulfide and nitrate reached 99.0% and 93.9%, and the production of elemental sulfur and nitrogen reached 63.1 mg and 79.5 mL, respectively.

DL-cysteine and L-cystine formation and their enhancement effects during sulfur autotrophic denitrification.

Sulfur autotrophic denitrification has been proved feasible for nitrate removal from aquatic environments and it utilizes elemental sulfur as the electron donor. A maximum denitrification rate of 194.57 mg N/L·d was achieved with biogenic sulfur as electron donor in a mixed culture collected from sulfur packed bed reactors; this rate was considerably higher than that delivered by α-S8 or µ-S in the same mixed culture. The elemental sulfur was also tested in the pure culture of Thiobacillus denitrificans, while a lower denitrification rate was noted than in the mixed culture, bio-S (4.86 mg N/L·d) again outperformed other two elemental sulfur's. X-ray absorption near edge structure spectra were collected to examine possible metabolic intermediates during the sulfur autotrophic denitrification process. The analysis revealed the existence of two major intermediates: DL-cysteine and L-cystine. They were found to not only provide electrons but also play a critical role in promoting the elemental sulfur-mediated sulfur autotrophic denitrification process. In general, we investigated the formation and enhancement effects of sulfur intermediates in the sulfur autotrophic denitrification process.

Characterisation of thiocyanate degradation in a mixed culture activated sludge process treating coke wastewater.

Microbial degradation of thiocyanate (SCN-) has been reported to suffer from instability highlighting the need for improved understanding of underlying mechanisms and boundaries. Respirometry, batch tests and DNA sequencing analysis were used to improve understanding of a mixed culture treating coke wastewater rich in SCN-. An uncultured species of Thiobacillus was the most abundant species (26%) and displayed similar metabolic capabilities to Thiobacillus denitrificans and Thiobacillus thioparus. Thiocyanate was hydrolysed/oxidised to NH4+-N, HCO3- and SO42-. Nevertheless, at 360-2100 mg SCN-/L a breakdown in the degradation pathway was observed. Respirometry tests demonstrated that NH4+-N was inhibitory to SCN- degradation (IC50: 316 mg/L). Likewise, phenol (180 mg/L) and hydroxylamine (0.25-16 mg/L) reduced SCN- degradation by 41% and ca. 7%, respectively. The understanding of the SCN- degradation pathways can enable stable treatment efficiencies and compliance with effluent of <4 mg SCN/L, required by the Industrial Emissions Directive.

Biological oxidation of iron sulfides.

The biological oxidation of minerals and ores, called bioleaching, has been studied for the last decades to solubilize metals and recover them. In particular, iron sulfides are the most studied ores for an optimum extraction of different metals, such as copper or zinc. The use of chemolithotrophic bacteria, as Acidothiobacillus ferrooxidans, to oxidize both iron and sulfur species in aerobic conditions and at acidic pH shows promising results. In the field of heritage preservation, the development of "green" treatments is more and more studied. Waterlogged archeological wood presents an accumulation of iron sulfides within its structure, which, after exposition to oxygen, lead to salt precipitation and acidification and so to the degradation of the wooden artifact. A new extraction method, based on the dissolution of iron sulfides by the use of bacteria could be an alternative to the current chemical extraction methods, as being more respectful and ecological. While A. ferrooxidans is very effective in mines and groundwater, in the field of conservation-restoration of wood, Thiobacillus denitrificans is a better candidate as it grows at neutral pH, which is less aggressive for organic substrates (wood here). Preliminary studies show the efficiency of T. denitrificans for the dissolution of iron sulfides, as the concentration of nitrates used as electron donors decreases while the concentration of sulfates produced increases without degrading the wooden matrix. Long-term behavior should be studied to assess the stability of the artifacts after treatment.

High nitrate removal by starch-stabilized Fe0 nanoparticles in aqueous solution in a controlled system.

This study was conducted to investigate biodenitrification efficiency with starch-stabilized nano zero valent iron (S-nZVI) as the additional electron donor in the presence of S2O3 in aqueous solutions, under anaerobic conditions. The main challenge for nZVI application is their tendency to agglomeration, thereby resulting in loss of reactivity that necessitates the use of stabilizers to improve their stability. In this study, S-nZVI was synthesized by chemical reduction method with starch as a stabilizer. The synthesized nanoparticles were characterized by TEM, XRD, and FTIR. Transmission electron microscopy (TEM) image shows S-nZVI has a size in the range of 5-27.5 nanometer. Temperature and S-nZVI concentration were the important factors affecting nitrate removal. Biodenitrification increased at 35°C and 500 mg/L of S-nZVI, in these conditions, biodenitrification efficiency increased from 40.45 to 78.84%. Experimental results suggested that biodenitrification increased by decreasing initial nitrate concentration. In the bioreactor biodenitrification rate was 94.07% in the presence of S-nZVI. This study indicated that, Fe2+ could be used as the only electron donor or as the additional electron donor in the presence of S2O3 to increase denitrification efficiency.

Identify biosorption effects of Thiobacillus towards perfluorooctanoic acid (PFOA): Pilot study from field to laboratory.

The concentration of Perfluoroalkyl acids (PFAAs) and the bacterial community composition along the Xiaoqing River were explored with HPLC-MS/MS and Illumina high-throughput sequencing in present study. The results showed that perfluorooctanoic acid (PFOA) was the predominant PFAAs in all sediment samples, and high level of PFOA could lead to an evident increase in the abundance of Thiobacillus. Thiobacillus was identified with the survival ability in high concentrations of PFOA accordingly. Therefore, Thiobacillus thioparus and Thiobacillus denitrificans were selected as receptors to design indoor biosorption experiment. The growth curves under different PFOA concentrations and residual rates of PFOA in the processes of cultivation were analyzed. The results showed that upwards concentrations of PFOA below 5000 ng/L led to an obvious increase in the growth rate of T. thioparus. Whereas PFOA promoted the growth of T. denitrificans in a relatively limited range of concentration, and the effect was not obvious. The addition of different concentrations of PFOA had no apparent effects on pH values in the media of both T. thioparus and T. denitrificans. The concentrations of PFOA in liquid media reduced after the process of bacteria culturing. The removal rates of T. thioparus and T. denitrificans to PFOA were 21.1-26.8% and 13.5-18.4%, respectively. The current findings indicated that T. thioparus could play a significant role as potential biosorbent with the ability to eliminate PFOA effectively in aquatic environment, which would provide novel information for PFOA ecological decontamination and remediation.

Enhanced fatty acid production in engineered chemolithoautotrophic bacteria using reduced sulfur compounds as energy sources.

Chemolithoautotrophic bacteria that oxidize reduced sulfur compounds, such as H2S, while fixing CO2 are an untapped source of renewable bioproducts from sulfide-laden waste, such as municipal wastewater. In this study, we report engineering of the chemolithoautotrophic bacterium Thiobacillus denitrificans to produce up to 52-fold more fatty acids than the wild-type strain when grown with thiosulfate and CO2. A modified thioesterase gene from E. coli ('tesA) was integrated into the T. denitrificans chromosome under the control of Pkan or one of two native T. denitrificans promoters. The relative strength of the two native promoters as assessed by fatty acid production in engineered strains was very similar to that assessed by expression of the cognate genes in the wild-type strain. This proof-of-principle study suggests that engineering sulfide-oxidizing chemolithoautotrophic bacteria to overproduce fatty acid-derived products merits consideration as a technology that could simultaneously produce renewable fuels/chemicals as well as cost-effectively remediate sulfide-contaminated wastewater.

Role of humic substances in promoting autotrophic growth in nitrate-dependent iron-oxidizing bacteria.

Nitrate-dependent iron oxidation was discovered in 1996 and has been reported from various environments ever since. To date, despite the widespread nature of this process, all attempts to cultivate chemolithoautotrophic nitrate-dependent iron oxidizers have been unsuccessful. The present study was focused on understanding the influence of natural chelating agents of iron, like humic substances, on the culturability, activity, and enumeration, of these microorganisms. Pure culture studies conducted with Thiobacillus denitrificans showed a constant increase in cell mass with a corresponding nitrate-dependent iron oxidation activity only when Fe(II) was provided together with humic substances, compared to no growth in control incubations without humic substances. The presence of a relatively strong chelating agent, such as EDTA, inhibited the growth of Thiobacillus denitrificans. It was concluded that complex formation between humic substances and iron was required for chemolithoautotrophic nitrate-dependent iron oxidation. Most probable number enumerations showed that numbers of chemolithoautotrophic nitrate-dependent iron-oxidizing bacteria were one to three orders of magnitude higher in the presence of humic substances compared to media without. Similar results were obtained when potential nitrate-dependent iron oxidation activity was determined in soil samples. In summary, this study showed that humic substances significantly enhanced the growth and activity of autotrophic nitrate-dependent iron-oxidizing microorganisms, probably by chelation of iron.

Genome-enabled studies of anaerobic, nitrate-dependent iron oxidation in the chemolithoautotrophic bacterium Thiobacillus denitrificans.

Thiobacillus denitrificans is a chemolithoautotrophic bacterium capable of anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation, both of which can strongly influence the long-term efficacy of in situ reductive immobilization of uranium in contaminated aquifers. We previously identified two c-type cytochromes involved in nitrate-dependent U(IV) oxidation in T. denitrificans and hypothesized that c-type cytochromes would also catalyze Fe(II) oxidation, as they have been found to play this role in anaerobic phototrophic Fe(II)-oxidizing bacteria. Here we report on efforts to identify genes associated with nitrate-dependent Fe(II) oxidation, namely (a) whole-genome transcriptional studies [using FeCO3, Fe(2) (+), and U(IV) oxides as electron donors under denitrifying conditions], (b) Fe(II) oxidation assays performed with knockout mutants targeting primarily highly expressed or upregulated c-type cytochromes, and (c) random transposon-mutagenesis studies with screening for Fe(II) oxidation. Assays of mutants for 26 target genes, most of which were c-type cytochromes, indicated that none of the mutants tested were significantly defective in nitrate-dependent Fe(II) oxidation. The non-defective mutants included the c 1-cytochrome subunit of the cytochrome bc 1 complex (complex III), which has relevance to a previously proposed role for this complex in nitrate-dependent Fe(II) oxidation and to current concepts of reverse electron transfer. A transposon mutant with a disrupted gene associated with NADH:ubiquinone oxidoreductase (complex I) was ~35% defective relative to the wild-type strain; this strain was similarly defective in nitrate reduction with thiosulfate as the electron donor. Overall, our results indicate that nitrate-dependent Fe(II) oxidation in T. denitrificans is not catalyzed by the same c-type cytochromes involved in U(IV) oxidation, nor have other c-type cytochromes yet been implicated in the process.