Iron Reduction and Soil Phosphorus Solubilization in Humid Tropical Forests Soils: The Roles of Labile Carbon Pools and an Electron Shuttle Compound

The affinity of iron oxides and hydroxides for phosphorus is thought to contribute to phosphorus limitation to net primary productivity in humid tropical forests on acidic, highly weathered soils. Perennially warm, humid conditions and high biological activity in these soils can result in fluctuating redox potential that in turn leads to considerable iron reduction in the presence of labile carbon and humic substances. We investigated the effects of reducing conditions in combination with the addition of labile carbon substrates (glucose and acetate) and an electron shuttle compound on iron reduction and phosphorus release in a humid tropical forest soil. Glucose or acetate was added to soils as a single dose at the beginning of the experiment, and as pulsed inputs over time, which more closely mimics patterns in labile carbon availability. Iron reduction and phosphorus mobilization were weakly stimulated by a single low level addition of carbon, and the addition of the electron shuttle compound with or without added carbon. Pulsed labile carbon additions produced a significant increase in soil pH, soluble iron, and phosphorus concentrations. Pulsed labile carbon inputs also promoted the precipitation of ferrous hydroxide complexes which could increase the capacity for P sorption, although our results suggest that rates of P solubilization exceeded re-adsorption. Plant and microbial P demand are also likely to serve as an important sinks for released P, limiting the role of P re-adsorption. Our results suggest that reducing conditions coupled with periodic carbon inputs can stimulate iron reduction and a corresponding increase in soil phosphorus mobilization, which may provide a source of phosphorus to plants and microorganisms previously undocumented in these ecosystems.     

Effect of Iron Bioavailability on Dissolved Hydrogen Concentrations During Microbial Iron Reduction

Dissolved hydrogen (H2) concentrations have been shown to correlate with specific terminal electron accepting processes (TEAPs) in aquifers. The research presented herein examined the effect of iron bioavailability on H2 concentrations during iron reduction in flow-through column experiments filled with soil obtained from the uncontaminated background area of the Field Research Center (FRC), Oak Ridge, TN and amended with acetate as the electron donor. The first column experiment measured H2 concentrations over 500 days of column operation that fluctuated within a substantial range around an average of 3.9 nM. Iron reduction was determined to be the dominant electron accepting process. AQDS (9,10-anthraquinone-2,6-disulfonic acid) was then used to determine if H2 concentrations during iron reduction were related to iron bioavailability. For this purpose, a 100-day flow-through column experiment was conducted that compared the effect of AQDS on iron reduction and subsequent H2 concentrations using two columns in parallel. Both columns were packed with FRC soil and inoculated with Geobacter sulfurreducens but only one was supplied with AQDS. The addition of AQDS increased the rate of iron reduction in the flow-through column and slightly decreased the steady-state H2 concentrations from an average of 4.0 nM for the column without AQDS to 2.0 nM for the column with AQDS. The results of this study therefore show that H2 can be used as an indicator to monitor rate and bioavailability changes during microbial iron reduction.     

Quinones as terminal electron acceptors for anaerobic microbial oxidation of phenolic compounds

The capacity of anaerobic granular sludge for oxidizing phenoland p-cresol under anaerobic conditions was studied. Phenol and p-cresolwere completely converted to methane when bicarbonate was the only terminal electron acceptor available. When the humic model compound, anthraquinone-2,6-disulfonate, was included as an alternative electron acceptor in the cultures, the oxidation of the phenolic compounds was coupled to the reduction of the model humic compound to its corresponding hydroquinone, anthrahydroquinone-2,6-disulfonate. These results demonstrate for the first time that the anaerobic degradation of phenolic compounds can be coupled to the reduction of quinones as terminal electron acceptor.     

Competition between methanogenesis and quinone respiration for ecologically important substrates in anaerobic consortia

Anaerobic consortia obtained from a wide variety of environments were tested for oxidizing several ecologically significant substrates with the humic model compound, anthraquinone-2,6-disulfonate (AQDS), as terminal electron acceptor. All the substrates, including hydrogen, acetate, propionate, methanol and lactate, were completely or partially converted to methane when bicarbonate was the only electron acceptor available. Addition of AQDS (20 mM) to the cultures prevented methanogenesis in most cases and AQDS reduction became the preferred pathway. AQDS was shown to be toxic for methanogenesis and this effect played an important role in enabling quinone-respiring bacteria to outcompete methanogens. Furthermore, AQDS respiration is thermodynamically more favorable than methanogenesis. All the consortia evaluated were capable of oxidizing hydrogen linked to the reduction of AQDS. Most inocula tested were also able to oxidize acetate and lactate in the same way. When methanol was provided as an electron donor competition between methanogenesis and acetogenesis occurred. Acetate accumulated from the latter process was responsible for quinone respiration. These results suggest that quinone-respiring bacteria are ubiquitous and that quinones in humus may significantly contribute to carbon cycling process by serving as a terminal electron acceptor for the anaerobic microbial oxidation of a wide variety of ecologically important substrates.     

Effect of redox mediator, AQDS, on the decolourisation of a reactive azo dye containing triazine group in a thermophilic anaerobic EGSB reactor

The feasibility of thermophilic (55 °C) anaerobic treatment applied to colour removal of a triazine contained reactive azo dye was investigated in two 0.53 l expanded granular sludge blanket (EGSB) reactors in parallel at a hydraulic retention time (HRT) of 10 h. Generally, this group of azo dyes shows the lowest decolourisation rates during mesophilic anaerobic treatment. The impact of the redox mediator addition on colour removal rates was also evaluated. Reactive Red 2 (RR2) and anthraquinone-2,6-disulfonate (AQDS) were selected as model compounds for azo dye and redox mediator, respectively. The reactors achieved excellent colour removal efficiencies with a high stability, even when high loading rates of RR2 were applied (2.7 g RR2 l⁻¹ per day). Although AQDS addition at catalytic concentrations improved the decolourisation rates, the impact of AQDS on colour removal was less apparent than expected. Results show that the AQDS-free reactor R2 achieved excellent colour removal rates with efficiencies around 91%, compared with the efficiencies around 95% for the AQDS-supplied reactor R1. Batch experiments confirmed that the decolourisation rates were co-substrate dependent, in which the volatile fatty acids (VFA) mixture was the least efficient co-substrate. The highest decolourisation rate was achieved in the presence of either hydrogen or formate, although the presence of glucose had a significant impact on the colour removal rates.     

Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the redox active analogue for humic substances

The bacterium Geobacter sulfurreducens can transfer electrons to quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH₂QDS) can also be used as energy source by G. sulfurreducens. Such bidirectional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bifunctional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH₂QDS. Using stopped-flow kinetic measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for ¹³C,¹⁵N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and ¹H heme methyl signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens.     

Application of redox mediators to accelerate the transformation of reactive azo dyes in anaerobic bioreactors.

Azo dyes are nonspecifically reduced under anaerobic conditions but the slow rates at which reactive azo dyes are converted presents a serious problem for the application of anaerobic technology as a first stage in the complete biodegradation of these compounds. As quinones have been found to catalyze reductive transfers by acting as redox mediators, the application of anthraquinone-2,6-disulfonic acid (AQDS) during continuous anaerobic treatment of the reactive azo dye, Reactive Red 2 (RR2), was evaluated. A mixture of volatile fatty acids was used as the electron-donating primary substrate. Batch experiments demonstrated that AQDS could increase the first-order rate constant of RR2 reductive cleavage by one order of magnitude. In the continuous experiment, treatment of RR2 containing synthetic wastewater in a lab-scale upflow anaerobic sludge blanket (UASB) reactor yielded low dye removal efficiencies (<30%). Consequently, severe toxicity problems occurred, eventually resulting in almost complete inhibition of the methanogenic activity. Addition of catalytic concentrations of AQDS (19 microM) to the reactor influent caused an immediate increase in the dye removal efficiency and recovery of biological activity. Ultimately, RR2 removal efficiency stabilized at 88%, and higher AQDS loads resulted in higher RR2 removal efficiencies (up to 98% at 155 microM AQDS). Examination of the RR2 decolorizing properties of dye-adapted reactor sludge and of nonadapted reactor seed sludge revealed that RR2 decolorization was principally a biologically driven transfer of reducing equivalents from endogenous and added substrates to the dye. Hydrogen, added in bulk, was clearly the preferred electron donor. Bacteria that couple dye decolorization to hydrogen oxidation were naturally present in seed sludge. However, enrichment was required for the utilization of electrons from volatile fatty acids for dye reduction. The stimulatory effect of AQDS on RR2 decolorization by AQDS-unadapted sludge was mainly due to assisting the electron transfer from endogenous substrates in the sludge to the dye. The stimulatory effect of AQDS on RR2 decolorization by sludge from the AQDS-exposed reactor was, in addition, strongly associated with the transfer of electrons from hydrogen and acetate to the dye, probably due to enrichment of specialized AQDS-reducing bacteria.     

Effect of iron(III), humic acids and anthraquinone-2,6-disulfonate on biodegradation of cyclic nitramines by Clostridium sp. EDB2

AIMS: To determine the biodegradation of cyclic nitramines by an anaerobic marine bacterium, Clostridium sp. EDB2, in the presence of Fe(III), humic acids (HA) and anthraquinone-2,6-disulfonate (AQDS). METHODS AND RESULTS: An obligate anaerobic bacterium, Clostridium sp. EDB2, degraded RDX and HMX, and produced similar product distribution including nitrite, methylenedinitramine, nitrous oxide, ammonium, formaldehyde, formic acid and carbon dioxide. Carbon (C) and nitrogen (N) mass balance for RDX products were 87% and 82%, respectively, and for HMX were 88% and 74%, respectively. Bacterial growth and biodegradation of RDX and HMX were stimulated in the presence of Fe(III), HA and AQDS suggesting that strain EDB2 utilized Fe(III), HA and AQDS as redox mediators to transfer electrons to cyclic nitramines. CONCLUSIONS: Strain EDB2 demonstrated a multidimensional approach to degrade RDX and HMX: first, direct degradation of the chemicals; second, indirect degradation by reducing Fe(III) to produce reactive-Fe(II); third, indirect degradation by reducing HA and AQDS which act as electron shuttles to transfer electrons to the cyclic nitramines. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study could be helpful in determining the fate of cyclic nitramine energetic chemicals in the environments rich in Fe(III) and HA.