Naphthalene Mineralization Research Articles

Taxis-enhanced mineralization and co-metabolism of PAHs by bacteria in micrometer-scale environments

Polycyclic aromatic hydrocarbons (PAHs) are associated with micropores in sediments and soils. This limits the bioaccessibility of these compounds via existing bioremediation technologies, as biodegradation is strongly influenced by the ability of bacteria to access different sizes of pores. In this work, we employed naphthalene and pyrene as model contaminants to evaluate the transformation capacity of the soil bacterium Pseudomonas putida G7 (2 × 1 μm) via mineralization and co-metabolic activity, respectively. Under non-growing conditions and in the absence of hydraulic flow, we examined how the tactic behavior of this motile bacterium influenced biodegradation of these two PAHs when passing through membranes with micrometer-sized pores (3 and 5 μm). The bacteria were spontaneously retained by the membranes, which blocked the contaminants away from a passive dosing source. However, the cells were mobilized through 5 μm pores after the application plant root exudate components (γ-aminobutyric acid, citrate and fructose) as strong chemoeffectors, which enhanced the mineralization of naphthalene and co-metabolism of pyrene. The tactic-mediated biodegradation enhancement did not occur through 3 μm pores, possibly due a physical constrain to the gradient sensing mechanism. Our results suggest that bacterial transport by chemotaxis may enhance the biotransformation of poorly bioaccessible contaminants present in micro-meter scale environments.

Decomposition of Naphthalene by Dielectric Barrier Discharge in Conjunction with a Catalyst at Atmospheric Pressure

In this study, coaxial dielectric barrier discharge (DBD) plasma, in conjunction with a metal oxide catalyst, was used to degrade naphthalene. The characteristics of plasma discharge were studied by measuring voltage and current waveforms and the Lissajous figure. The effects of different parameters of the process on naphthalene decomposition in air were investigated. XRD, BET, and SEM data were used to investigate the nature, specific surface area, and surface morphology of the catalyst. The results show that the mineralization of naphthalene reached 82.2% when the initial naphthalene concentration was 21 ppm and the total gas flow rate was 1 L/min in the DBD reactor filled with Al2O3. The mineralization of naphthalene first increased and then became stable with the increase in treatment time and discharge power. The TiO2 catalyst has more apparent advantages than the two other studied catalysts in terms of the removal efficiency and mineralization of naphthalene due to this catalyst’s large specific surface area, porous structure, and photocatalytic properties. In addition, the introduction of a small amount of water vapor can promote the mineralization and CO2 selectivity of naphthalene. With further increases in the water vapor, Fe2O3 has a negative effect on the naphthalene oxidation due to its small pore size. The TiO2 catalyst can overcome the adverse effects of water molecule attachment due to its photocatalytic properties.

Naphthalene mineralization by supercritical water oxidation and determination of by-products using non-target analysis

Abstract In this study, the fate of naphthalene was investigated with a supercritical water oxidation process (SCWO). Also, the effectiveness of different operating conditions including pressure, temperature, residence time and oxidant dose on the formation of by-products was determined. The experimental sets were determined by the experimental design program and the effect of the selected variables on the removal of the naphthalene was associated statistically. According to obtained results, naphthalene by SCWO process was mineralized up to 98.5%. Removal efficiencies in sub- and supercritical conditions were between 94 and 100%. Derivatives of aldehyde, propanoic acid, benzene acetic acid and benzofuran were detected as by-products at many experimental conditions and also, some intermediates with a molecular weight higher than naphthalene were determined.

Bacterial Endophytes Isolated from Plants in Natural Oil Seep Soils with Chronic Hydrocarbon Contamination.

The bacterial endophytic communities of four plants growing abundantly in soils highly contaminated by hydrocarbons were analyzed through culturable and culture-independent means. Given their tolerance to the high levels of petroleum contamination at our study site, we sought evidence that Achillea millefolium, Solidago canadensis, Trifolium aureum, and Dactylis glomerata support high levels of hydrocarbon degrading endophytes. A total of 190 isolates were isolated from four plant species. The isolates were identified by partial 16S rDNA sequence analysis, with class Actinobacteria as the dominant group in all species except S. canadensis, which was dominated by Gammaproteobacteria. Microbacterium foliorum and Plantibacter flavus were present in all the plants, with M. foliorum showing predominance in D. glomerata and both endophytic bacterial species dominated T. aureum. More than 50% of the isolates demonstrated degradative capabilities for octanol, toluene, naphthalene, kerosene, or motor oil based on sole carbon source growth screens involving the reduction of tetrazolium dye. P. flavus isolates from all the sampled plants showed growth on all the petroleum hydrocarbons (PHCs) substrates tested. Mineralization of toluene and naphthalene was confirmed using gas-chromatography. 16S based terminal restriction fragment length polymorphism analysis revealed significant differences between the endophytic bacterial communities showing them to be plant host specific at this site. To our knowledge, this is the first account of the degradation potential of bacterial endophytes in these commonly occurring pioneer plants that were not previously known as phytoremediating plants.

Gamma radiolytic degradation of naphthalene in aqueous solution

Abstract The decomposition of naphthalene in aqueous solution was studied using gamma irradiation combined with both H2O2 and TiO2 nanoparticles. Gamma irradiation led to a complete degradation of naphthalene and a partial mineralization. With initial concentration of 5–32 mg/L, more than 98% of naphthalene was removed and TOC reduction reached 28–31% at an absorbed dose of 3.0 kGy. The degradation of naphthalene was faster at neutral pH and the initial degradation rate increased with increasing the initial concentration of naphthalene. Addition of H2O2 and TiO2 nanoparticles all enhanced the degradation and mineralization of naphthalene. TOC removal efficiency increased from 28% (irradiation alone) to 35% with addition of H2O2 (40 mg/L), and to 48% with addition of TiO2 (0.8 g/L). The degradation of naphthalene in aqueous solution by gamma irradiation was mainly through the oxidation by ·OH radicals. The intermediate naphthol and carboxylic acids such as formic acid and oxalic acid were identified by LC–MS and IC.

Anaerobic naphthalene degradation by sulfate-reducing Desulfobacteraceae from various anoxic aquifers.

Polycyclic aromatic hydrocarbons (PAH) are widespread and persistent environmental contaminants, especially in oxygen-free environments. The occurrence of anaerobic PAH-degrading bacteria and their underlying metabolic pathways are rarely known. In this study, PAH degraders were enriched in laboratory microcosms under sulfate-reducing conditions using groundwater and sediment samples from four PAH-contaminated aquifers. Five enrichment cultures were obtained showing sulfate-dependent naphthalene degradation. Mineralization of naphthalene was demonstrated by the formation of sulfide concomitant with the depletion of naphthalene and the development of (13)C-labeled CO2 from [(13)C6]-naphthalene. 16S rRNA gene and metaproteome analyses revealed that organisms related to Desulfobacterium str. N47 were the main naphthalene degraders in four enrichment cultures. Protein sequences highly similar to enzymes of the naphthalene degradation pathway of N47 were identified, suggesting that naphthalene was activated by a carboxylase, and that the central metabolite 2-naphthoyl-CoA was further reduced by two reductases. The data indicate an importance of members of the family Desulfobacteraceae for naphthalene degradation under sulfate-reducing conditions in freshwater environments.

Physiological role of the novel salicylaldehyde dehydrogenase NahV in mineralization of naphthalene by Pseudomonas putida ND6

The classical salicylaldehyde dehydrogenases found in naphthalene-degrading bacteria are denoted as NahF. In addition to NahF, NahV, and its corresponding gene nahV, were found here in multiple naphthalene-degrading bacteria isolated from industrial wastewater polluted with polycyclic aromatic hydrocarbons (PAHs). In this study, we described for the first time the biological function and regulation model of NahV for the mineralization of naphthalene by P. putida ND6 via the construction of nahF-, nahV- and regulatory gene nahR-deficient strains. The two mutants of salicylaldehyde dehydrogenase genes and wild-type Pseudomonas ND6 were compared with respect to growth rate, naphthalene degradation efficiency, protein expression level, and salicylaldehyde dehydrogenase activity. The data showed that the presence of NahV conferred a physiological advantage on P. putida ND6 for the catabolism of naphthalene in the presence of NahF. NahV could facilitate naphthalene degradation by increasing total salicylaldehyde dehydrogenase activity when both dehydrogenases are present and it could replace the function of NahF when nahF gene is deleted or mutated, thus ensuring mutants could survive in naphthalene-containing environments. To investigate regulation model of NahV, we detected the expression levels and salicylaldehyde dehydrogenase activity in the wild-type and the nahR mutant strains following cultivation in the presence of glucose±salicylate. The data demonstrated that just like the classical salicylaldehyde dehydrogenases, NahF, NahV was induced by salicylate in the presence of NahR.

Meiofauna reduces bacterial mineralization of naphthalene in marine sediment

The role of sediment-living meiofauna, benthic invertebrates smaller than 1000 μm such as nematodes and ostracods, on the mineralization of naphthalene, a common polycyclic aromatic hydrocarbon (PAH) in marine sediments, was studied in microcosms using radiorespirometry. A method to extract live meiofauna was developed and used in order to experimentally manipulate meiofauna abundance and group diversity. Higher abundances of meiofauna were found to significantly decrease naphthalene mineralization. Furthermore, a change in the bacterial community composition (studied using terminal restriction fragment length polymorphism) was also observed in presence of higher meiofauna abundance, as well as a lower number of cultivable naphthalene-degrading bacteria. The reduced mineralization of naphthalene and the altered bacterial community composition in the presence of increased meiofauna abundance is likely the result of top-down control by meiofauna. This study shows that higher abundances of meiofauna can significantly decrease the microbial mineralization of PAHs such as naphthalene and also significantly modify the bacterial community composition in natural marine sediments.

Bioremediation of naphthalene in water by Sphingomonas paucimobilis using new biodegradable surfactants based on poly (ɛ-caprolactone)

New amphiphilic block surfactants ABA based on a central segment of polycaprolactone with different molecular composition were evaluated in the bioremediation of naphthalene in water by Sphingomonas paucimobilis and compared with sodium dodecyl sulphate as reference surfactant (SDS). Also the biodegradation of the new surfactants by bacteria, S. paucimobilis and a mixture of bacteria ( Pseudomonas aureginosa, Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus megaterium) was studied by indirect impedance technique and carbon dioxide determination. All the bacteria biodegraded in solution and micellar phase the central segment of PCL with mineralization rates in the range of 0.024–0.036 mg of CO 2 per day. S. paucimobilis biodegraded naphthalene in the presence of the new surfactants and GC analysis demonstrated that conversion to products started immediately after inoculum. In all the experiments, except for SDS, at 140 h of incubation time, the remaining naphthalene concentration was about 10% of the initial concentration. In contrast, the production of CO 2 was delayed 4–7 days and values around 75% of naphthalene mineralization degree were achieved in three weeks. The addition of PCL-surfactants, in solution and in micellar phase, not interfered in the naphthalene mineralization. These results have shown promising potential of these biodegradable PCL-surfactants in surfactant-enhanced remediation (SER) technology for removing residual organics from contaminated groundwater and soils.

Functional Gene Abundances (nahAc, alkB, xylE) in the Assessment of the Efficacy of Bioremediation

In this study, we compared the mineralization rates of three selected (14)C-labeled hydrocarbon compounds, octacosane, toluene, and naphthalene, with the presence of the corresponding functional genes (alkB, xylE, nahAc) in a large number of soil samples representing different types of soil and petroleum hydrocarbon contamination. Functional genes were enumerated by the replicate limited dilution (RLD) polymerase chain reaction (PCR) technique. RLD-PCR was further compared to real-time PCR measurements for nahAc and xylE for some samples. At a heating oil-contaminated site, octacosane mineralization rates were higher (on average 0.0015 day(-1)) when compared to aerobic naphthalene and toluene mineralization (on average 0.00003 and 0.0007 day(-1)). The corresponding gene abundances measured by RLD-PCR were on average 0.95, 0.3, and 0.13 x 10(3) gene copies g(-1) soil for alkB, nahAc, and xylE, respectively. At a site contaminated with gasoline, the situation was the opposite: Toluene mineralization was the highest (on average 0.0031 day(-1)), and only xylE genes could be detected (on average 0.13 x 10(3) gene copies g(-1) soil by RLD-PCR). XylE and nahAc gene abundances were correlated with the (14)C-toluene and naphthalene mineralization activities, respectively, in samples from aerobic layers. AlkB gene abundances were not correlated with the octacosane mineralization. Real-time PCR was a more sensitive method than RLD-PCR by a factor of 1,200 for nahAc and 300 for xylE. In conclusion, functional gene abundances seemed to reflect the type of the contamination. With optimized assays, the gene abundances can be used to assess bioremediation efficacy.

Evaluating the biodegradation of aromatic hydrocarbons by monitoring of several functional genes

Various microbial activities determine the effectiveness of bioremediation processes. In this work, we evaluated the feasibility of gene array hybridization for monitoring the efficiency of biodegradation processes. Biodegradation of 14C-labelled naphthalene and toluene by the aromatic hydrocarbon-degrading Pseudomonas putida F1, P. putida mt-2 and P. putida G7 was followed in mixed liquid culture microcosm by a preliminary, nylon membrane-based gene array. In the beginning of the study, toluene was degraded rapidly and increased amount of toluene degradation genes was detected by the preliminary gene array developed for the study. After toluene was degraded, naphthalene mineralization started and the amount of naphthalene degradation genes increased as biodegradation proceeded. The amount of toluene degradation genes decreased towards the end of the study. The hybridization signal intensities determined by preliminary gene array were in good agreement with mineralization of naphthalene and toluene and with the amount of naphthalene dioxygenase and toluene dioxygenase genes quantified by dot blot hybridization. The clear correlation between the results obtained by the preliminary array and the biodegradation process suggests that gene array methods can be considered as a promising tool for monitoring the efficiency of biodegradation processes.

Effects of Enrichment with Salicylate on Bacterial Selection and PAH Mineralization in a Microbial Community from a Bioreactor Treating Contaminated Soil

We investigated enrichment with salicylate as a method to stimulate the degradation of polycyclic aromatic hydrocarbons (PAHs) by a microbial communityfrom a bioreactortreating PAH-contaminated soil. DNA-based stable isotope probing (SIP) was used to compare the effect of alternate methods of salicylate addition (spike vs slow, continuous addition) on the diversity of the enriched microbial community. After identification of salicylate degraders by SIP, real-time quantitative PCR (qPCR) primers were developed to quantify the abundances of three groups containing salicylate-utilizing organisms in the bioreactor community before and after enrichment. The different methods of salicylate addition were found to select for different microbial communities. Two groups containing salicylate-degrading bacteria increased in abundance substantially after enrichment by continuous addition of salicylate but did not increase in abundance in response to the spike addition, whereas a third group increased in abundance in response to both methods of salicylate addition. The initial rate of naphthalene mineralization increased significantly after enrichment by spike addition of salicylate, but neither phenanthrene nor benzo[a] pyrene mineralization rates were enhanced. Continuous addition of salicylate did not enhance the mineralization rate for any of the PAHs. These results suggest that enrichment with salicylate can select for naphthalene-degrading bacteria, but does not select for organisms responsible for degrading PAHs of higher molecular weight. Differences in microbial selection observed in this study that resulted from different rates of carbon source addition also have implications for the design of SIP experiments with water-soluble carbon sources.

Mineralization of PAHs in coal–tar impacted aquifer sediments and associated microbial community structure investigated with FISH

The microbial community structure and mineralization of polycyclic aromatic hydrocarbons (PAHs) in a coal–tar contaminated aquifer were investigated spatially using fluorescence in situ hybridization (FISH) and in laboratory-scale incubations of the aquifer sediments. DAPI-detected microbial populations in the contaminated sediments were three orders of magnitude greater than nearby uncontaminated sediments, suggesting growth on coal–tar constituents in situ. Actinobacteria, β- and γ- Proteobacteria, and Flavobacteria dominated the in situ aerobic (>1 mg l −1 dissolved oxygen) microbial community, whereas sulfate-reducing bacteria comprised 37% of the microbial community in the sulfidogenic region of the aquifer. Rapid mineralization of naphthalene and phenanthrene were observed in aerobic laboratory microcosms and resulted in significant enrichment of β- and γ- Proteobacteria potentially explaining their elevated presence in situ. Firmicutes, Flavobacteria, α- Proteobacteria, and Actinobacteria were also enriched in the mineralization assays, but to a lesser degree. Nitrate- and sulfate-limited mineralization of naphthalene in laboratory microcosms occurred to a small degree in aquifer sediments from locations where groundwater chemistry indicated nitrate- and sulfate-reduction, respectively. Some iron-limited mineralization of naphthalene and phenanthrene was also observed in sediments originating near groundwater measurements of elevated ferrous iron. The results of this study suggest that FISH may be a useful tool for providing a much needed link between laboratory microcosms and groundwater measurements made in situ necessary to better demonstrate the potential for natural attenuation at complex PAH contaminated sites.

Molecular cloning of genenahH encoding extradiol-type dioxygenase from the NAH plasmid ofPseudomonas stutzeri NA1

Naphthalene-degradingPseudomonas stutzeri NA1 was found to harbour the NAH plasmid, which contains the classical upper and lower catabolic genes required for naphthalene mineralization. The lower pathway inP. stutzeri NA1 was found to proceedviameta-ring cleavage of catechol due to the presence of thenahH gene encoding extradiol catechol 2,3-dioxygenase. Naphthalene-induced cells were able to mineralise both salicylate and catechol. Absorption spectra and gas chromatography/mass spectrometry analysis ofritermediate metabolites of salicylate or catechol degradation by a crude extract ofP. stutzeri NA1 revealed the presence of themeta-ring cleavage product 2-hydroxymuconate semialdehyde as a major constituent. The extradiol ring cleavage genenahH was amplified successfully from the NAH plasmid ofP. stutzeri NA1 with catechol 2,3-dioxygenase-specific primers and cloned inEscherichia coli JM109 The complete nucleotide sequence of cloned PCR fragment was determined. Sequence analysis of cloned PCR fragment revealed an open reading frame with similarity to other extradiol dioxygenases. The deduced amino acid sequence ofnahH fromP. stutzeri NA1 showed 96% sequence identity with the catechol 2,3-dioxygenase gene fromPseudomonas putida strain H. However, when compared to othernahH genes from different pseudomonads, it was in a separate phylogenetic branch, indicating a degree of speciation among the extradiol dioxygenase family.

Protozoan Grazing Increases Mineralization of Naphthalene in Marine Sediment

Bacterial decomposition of organic matter is frequently enhanced when protozoa are present. Various mechanisms have been proposed to account for this phenomenon, including effects associated with grazing by protozoa (such as increased recycling of limiting nutrients, removal of senescent cells, or reduction of competition among bacteria) and indirect effects of grazers (such as excretion of bacterial growth factors). Few studies have examined the role of protozoa in bacterial degradation of xenobiotic compounds in sediment containing a natural community of microbes. The effect of protozoa on mineralization of naphthalene was investigated in this study. Laboratory experiments were conducted using field-contaminated estuarine sediment, with the indigenous microbial populations. Mineralization of naphthalene was up to four times greater in treatments with actively grazing protozoa than in treatments containing the grazing inhibitor cytochalasin B. Control experiments confirmed that the grazing inhibitor was not toxic to ciliates but did prevent them from grazing. The grazing inhibitor did not affect growth rates of a mixed culture of sediment bacteria or a pure polycyclic-aromatic-hydrocarbon-degrading strain. Once grazing had been inhibited, supplementing treatments with inorganic N and P, glucose, or additional protozoa failed to stimulate naphthalene mineralization. Naphthalene-degrading bacteria were four to nine times less abundant when protozoan grazing was suppressed. We suggest that protozoa enhance naphthalene mineralization by selectively grazing on those sediment bacteria that ordinarily would outcompete naphthalene-degrading bacteria.

Effects of naphthalene on microbial community composition in the Delaware estuary

The effects of naphthalene on microbial communities in the bottom boundary layer of the Delaware Bay estuary were investigated in microcosms using denaturing gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization (FISH) with oligonucleotide probes. Three days after the addition of naphthalene, rates of bacterial production and naphthalene mineralization were higher than in no-addition controls and than in cases where glucose was added. Analyses using both DGGE and FISH indicated that the bacterial community changed in response to the addition of naphthalene. FISH data indicated that a few major phylogenetic groups increased in response to the glucose addition and especially to the naphthalene addition. DGGE also demonstrated differences in community composition among treatments, with four phylotypes being unique to naphthalene-amended treatments and three of these having 16S rRNA genes similar to known hydrocarbon degraders. The bacterial community in the naphthalene-amended treatment was distinct from the communities in the glucose-amended treatment and in the no-addition control. These data suggest that polycyclic aromatic hydrocarbons may have large effects on microbial community structure in estuaries and probably on microbially mediated biogeochemical processes.

Naphthalene mineralization coupled to sulfate reduction in aquifer-derived enrichments

Naphthalene was microbially transformed in sulfate-reducing laboratory microcosms established under strictly anaerobic conditions using sediment from two sulfate-rich, coal tar-contaminated aquifers and enriched over a 3-year period. As much as 66% of [14C]naphthalene was mineralized to CO2 over 13 days. Addition of sodium molybdate inhibited sulfidogenesis and resulted in a 44% reduction in total [14C]naphthalene mineralized. Methane was never detected in active naphthalene-degrading microcosms, and formation of sulfide during naphthalene degradation was confirmed using a 35SO2−4 radiotracer technique. GC/MS analyses of stabilized naphthalene-degrading consortia indicated the formation of naphthalenol as a potential metabolic intermediate. These results demonstrate that naphthalene oxidation may be coupled to sulfate reduction in aquifer-derived sediments.

Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry

The effect of microbial inoculation on the mineralization of naphthalene in a bioslurry treatment was evaluated in soil slurry microcosms. Inoculation by Pseudomonas putida G7 carrying the naphthalene dioxygenase (nahA) gene resulted in rapid mineralization of naphthalene, whereas indigenous microorganisms in the PAH-contaminated soil required a 28 h adaptation period before significant mineralization occurred. The number of nahA-like gene copies increased in both the inoculated and non-inoculated soil as mineralization proceeded, indicating selection towards naphthalene dioxygenase producing bacteria in the microbial community. In addition, 16S rRNA analysis by denaturing gradient gel electrophoresis (DGGE) analysis showed that significant selection occurred in the microbial community as a result of biodegradation. However, the indigenous soil bacteria were not able to compete with the P. putida G7 inoculum adapted to naphthalene biodegradation, even though the soil microbial community slightly suppressed naphthalene mineralization by P. putida G7.

Degradation of the polycyclic aromatic hydrocarbon (PAH) fluorene is retarded in a Scots pine ectomycorrhizosphere.

• Polycyclic aromatic hydrocarbons (PAHs) are an important class of persistent organic pollutants (POPs) in the environment and accumulate in forest soils. These soils are often dominated by ectomycorrhizal (EcM) roots, but little is known about how EcM fungi degrade PAHs, or the overall effect of field colonized EcM roots on the fate of PAHs. • The ability of eight EcM fungi to degrade PAHs in liquid culture spiked with 14 C labelled PAHs was investigated. Microcosms were used to determine the impact of naturally colonized mycorrhizal pine seedlings on PAH mineralization and volatilization. • Only two EcM fungi (Thelephora terrestris and Laccaria laccata) degraded at least one PAH and none were able to mineralize the PAHs in pure culture. Where degradation occurred, the compounds were only mono-oxygenated. EcM pine seedlings did not alter naphthalene mineralization or volatilization but retarded fluorene mineralization by 35% compared with unplanted, ectomycorrhizosphere soil inoculated, microcosms. • The EcM fungi possessed limited PAH degrading abilities, which may explain why EcM dominated microcosms retarded fluorene mineralization. This observation is considered in relation to the 'Gadgil-effect', where retarded litter decomposition has been observed in the presence of EcM roots.

Nitrilotriacetate stimulation of anaerobic Fe(III) respiration by mobilization of humic materials in soil.

An enrichment culture capable of naphthalene mineralization reduced Fe(III) oxides without direct contact in anaerobic soil microcosms when the Fe(III) was placed in dialysis membranes or entrapped within alginate beads. Both techniques demonstrated that a component in soil, possibly humic materials, facilitated Fe(III) reduction when direct contact between cells and Fe(III) was not possible. The addition of the synthetic Fe(III) chelator, nitrilotriacetic acid (NTA), to soil enhanced Fe(III) reduction across the dialysis membrane and alginate beads, with the medium changing from clear to a dark brown color. An NTA-soil extract was more effective in Fe(III) reduction than the extracted soil itself. Characteristics of the NTA extract were consistent with that of humic substances. The results indicate that NTA improved Fe(III) reduction not by Fe(III) solubilization but by extraction of humic substances from soil into the aqueous medium. This is the first study in which stimulation of Fe(III) reduction through the addition of chemical chelators is shown to be due to the extraction of electron-shuttling compounds from the soil and not to solubilization of the Fe(III) and indicates that mobilization of humic materials could be an important component of anaerobic biostimulation.