Formulation of the CBC-Model for Modelling the Contaminants and Footprints in Natural Attenuation of BTEX

This paper provides the details of the Coupled Biological and Chemical (CBC) model for representing in situ bioremediation of BTEX. The CBC model contains novel features that allow it to comprehensively track the footprints of BTEX bioremediation, even when the fate of those footprints is confounded by abiotic reactions and complex interactions among different kinds of microorganisms. To achieve this comprehensive tracking of all the footprints, the CBC model contains important new biological features and key abiotic reactions. The biological module of the CBC-model includes these important new aspects: (1) it separates BTEX fermentation from methanogenesis, (2) it explicitly includes biomass as a sink for electrons and carbon, (3) it has different growth rates for each biomass type, and (4) it includes inhibition of the different reactions by other electron acceptors and by sulfide toxicants. The chemical module of the CBC-model includes abiotic reactions that affect the footprints of the biological reactions. In particular, the chemical module describes the precipitation/dissolution of CaCO3, Fe2O3, FeS, FeS2, and S degrees. The kinetics for the precipitation/dissolution reactions follow the critical review in Maurer & Rittmann (2004).     

Syntrophic Effects in a Subsurface Clostridial Consortium on Fe(III)-(Oxyhydr)oxide Reduction and Secondary Mineralization

In this study, we cultivated from subsurface sediments an anaerobic clostridial consortium that was composed of a fermentative Fe-reducer Clostridium species (designated as strain FGH) and a novel sulfate-reducing bacterium belonging to the clostridia family Vellionellaceae (designated as strain RU4). In pure culture, Clostridium sp. strain FGH mediated the reductive dissolution/transformation of iron oxides during growth on peptone. When Clostridium sp. FGH was grown with strain RU4 on peptone, the rates of iron oxide reduction were significantly higher. Iron reduction by the consortium was mediated by multiple mechanisms, including biotic reduction by Clostridium sp. FGH and biotic/abiotic reactions involving biogenic sulfide formed by strain RU4. The Clostridium sp. FGH produced hydrogen during fermentation, and the presence of hydrogen inhibited growth and iron reduction activity. The sulfate-reducing partner strain RU4 was stimulated by the presence of H₂and generated reactive sulfide which promoted the chemical reduction of the iron oxides. Characterization of Fe(II) mineral products showed the formation of nanoparticulate magnetite during ferrihydrite reduction, and the precipitation of iron sulfides during goethite and hematite reduction. The results suggest an important pathway for iron reduction and secondary mineralization by fermentative sulfate-reducing microbial consortia through syntrophy-driven biotic/abiotic reactions with biogenic sulfide.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Sulfur Species as Redox Partners and Electron Shuttles for Ferrihydrite Reduction by Sulfurospirillum deleyianum

Iron(III) (oxyhydr)oxides can represent the dominant microbial electron acceptors under anoxic conditions in many aquatic environments, which makes understanding the mechanisms and processes regulating their dissolution and transformation particularly important. In a previous laboratory-based study, it has been shown that 0.05 mM thiosulfate can reduce 6 mM ferrihydrite indirectly via enzymatic reduction of thiosulfate to sulfide by the sulfur-reducing bacterium Sulfurospirillum deleyianum, followed by abiotic reduction of ferrihydrite coupled to reoxidation of sulfide. Thiosulfate, elemental sulfur, and polysulfides were proposed as reoxidized sulfur species functioning as electron shuttles. However, the exact electron transfer pathway remained unknown. Here, we present a detailed analysis of the sulfur species involved. Apart from thiosulfate, substoichiometric amounts of sulfite, tetrathionate, sulfide, or polysulfides also initiated ferrihydrite reduction. The portion of thiosulfate produced during abiotic ferrihydrite-dependent reoxidation of sulfide was about 10% of the total sulfur at maximum. The main abiotic oxidation product was elemental sulfur attached to the iron mineral surface, which indicates that direct contact between microorganisms and ferrihydrite is necessary to maintain the iron reduction process. Polysulfides were not detected in the liquid phase. Minor amounts were found associated either with microorganisms or the mineral phase. The abiotic oxidation of sulfide in the reaction with ferrihydrite was identified as rate determining. Cysteine, added as a sulfur source and a reducing agent, also led to abiotic ferrihydrite reduction and therefore should be eliminated when sulfur redox reactions are investigated. Overall, we could demonstrate the large impact of intermediate sulfur species on biogeochemical iron transformations.     

Discrimination among iron sulfide species formed in microbial cultures

A quantitative method for the study of iron sulfides precipitated in liquid cultures of bacteria is described. This method can be used to quantify and discriminate among amorphous iron sulfide (FeS(amorph)), iron monosulfide minerals such as mackinawite or greigite (FeS(min)), and iron disulfide minerals such as pyrite or marcasite (FeS(2min)) formed in liquid cultures. Degradation of iron sulfides is performed using a modified Cr(2+) reduction method with reflux distillation. The basic steps of the method are: first, separation of FeS(amorph); second, elimination of interfering species of S such as colloidal sulfur (S(c) degrees ), thiosulphate (S(2)O(3)(2-)) and polysulfides (S(x)(2-)); third, separation of FeS(min); and fourth, separation of FeS(2min). The final product is H(2)S which is determined after trapping. The efficiency of recovery is 96-99% for FeS(amorph), 76-88% for FeS(min), and >97% for FeS(2min). This method has a high reproducibility if the experimental conditions are rigorously applied and only glass conduits are used. A well ventilated fume hood must be used because of the toxicity and volatility of several reagents and products. The advantage relative to previously described methods are better resolution for iron sulfide species and use of the same bottles for both incubation of cultures and acid degradation. The method can also be used for Fe/S stoichiometry with sub-sampling and Fe analysis.     

A new look at microbial leaching patterns on sulfide minerals

Leaching patterns on sulfide minerals were investigated by high-resolution scanning electron microscopy (SEM). Our goal was to evaluate the relative contributions of inorganic surface reactions and reactions localized by attached cells to surface morphology evolution. Experiments utilized pyrite (FeS(2)), marcasite (FeS(2)) and arsenopyrite (FeAsS), and two iron-oxidizing prokaryotes in order to determine the importance of cell type, crystal structure, and mineral dissolution rate in microbially induced pit formation. Pyrite surfaces were reacted with the iron-oxidizing bacterium Acidithiobacillus ferrooxidans (at 25 degrees C), the iron-oxidizing archaeon 'Ferroplasma acidarmanus' (at 37 degrees C), and abiotically in the presence of Fe(3+) ions. In all three experiments, discrete bacillus-sized (1-2 μm) and -shaped (elliptical) pits developed on pyrite surfaces within 1 week of reaction. Results show that attaching cells are not necessary for pit formation on pyrite. Marcasite and arsenopyrite surfaces were reacted with A. ferrooxidans (at 25 degrees C) and 'F. acidarmanus' (at 37 degrees C). Cell-sized and cell-shaped dissolution pits were not observed on marcasite or arsenopyrite at any point during reaction with A. ferrooxidans, or on marcasite surfaces reacted with 'F. acidarmanus'. However, individual 'F. acidarmanus' cells were found within individual shallow (<0.5 μm deep) pits. The size and shape (round rather than elliptical) of the pits conformed closely to the shape of F. acidarmanus (cells) pits on arsenopyrite. We infer these pits to be cell-induced. We attribute the formation of pits readily detectable (by SEM) to the higher reactivity of arsenopyrite compared to pyrite and marcasite under the conditions the experiment was conducted. These pits contributed little to the overall surface topographical evolution, and most likely did not significantly increase surface area during reaction. Our results suggest that overall sulfide mineral dissolution may be dominated by surface reactions with Fe(3+) rather than by reactions at the cell-mineral interface.     

Enhanced anaerobic biodegradation of BTEX-ethanol mixtures in aquifer columns amended with sulfate,chelated ferric iron or nitrate

Flow-through aquifer columns were used to investigate the feasibility of adding sulfate, EDTA–Fe(III) or nitrate to enhance the biodegradation of BTEX and ethanol mixtures. The rapid biodegradation of ethanol near the inlet depleted the influent dissolved oxygen (8 mg l^-1), stimulated methanogenesis, and decreased BTEX biodegradation efficiencies from >99% in the absence of ethanol to an average of 32% for benzene, 49% for toluene, 77% for ethylbenzene, and about 30% for xylenes. The addition of sulfate, EDTA–Fe(III) or nitrate suppressed methanogenesis and significantly increased BTEX biodegradation efficiencies. Nevertheless, occasional clogging was experienced by the column augmented with EDTA–Fe(III) due to iron precipitation. Enhanced benzene biodegradation (>70% in all biostimulated columns) is noteworthy because benzene is often recalcitrant under anaerobic conditions. Influent dissolved oxygen apparently played a critical role because no significant benzene biotransformation was observed after oxygen was purged out of the influent media. The addition of anaerobic electron acceptors could enhance BTEX biodegradation not only by facilitating their anaerobic biodegradation but also by accelerating the mineralization of ethanol or other substrates that are labile under anaerobic conditions. This would alleviate the biochemical oxygen demand (BOD) and increase the likelihood that entraining oxygen would be used for the biotransformation of residual BTEX.     

Observations on the diagenetic behavior of arsenic in a deep coastal sediment

Vertical profiles of total dissolved arsenic, manganese and iron, pH, Eh and rates of sulfate reduction were determined in a freshly-collected box core from a 335m depth station in the Laurentian Trough. The relationships observed between the profiles were further examined in the laboratory by measuring these same parameters with time in surficial sediment slurries as the Eh decreased in response to biological activity or chemical alteration.Both field and laboratory observations have shown that arsenic is released predominantly as As(III) into reducing sediment porewaters. This occurs after the dissolution of manganese oxides and at the same time as the dissolution of iron oxyhydroxides and the onset of sulfate reduction. Laboratory experiments indicated that sulfate reduction and the production of sulfide ions are not solely responsible for the release of arsenic to the porewaters, although this process is necessary to create and maintain a highly reducing environment conducive to rapid iron dissolution.The diagenesis of arsenic in Laurentain Trough sediments involves the simultaneous release of arsenic and iron at a subsurface depth, followed by its removal from porewaters by precipitation and adsorption reactions after migration by diffusion along concentration gradients. A qualitative model is presented to describe the behavior of arsenic in coastal marine sediments.Present address: Department of Geological Sciences, McGill University, 3450 UniversityStreet, Montreal, Quebec H3A 2A7, Canada     

Iron sulfide formation on root surfaces controlled by the life cycle of wild rice (<Emphasis Type="Italic">Zizania palustris</Emphasis>)

Iron sulfide plaques have been observed on roots of wild rice (Zizania palustris) and other wetland plants grown in sulfur-impacted freshwater ecosystems, but the mechanism of their formation and ramifications for plants have not been investigated. We exposed a model annual wetland plant, Zizania palustris, to elevated sulfate concentrations (3.1 mM) and quantified the development of iron oxide and iron sulfide precipitates on root surfaces throughout the plant life cycle. During the onset of seed production, root surfaces amended with sulfate transitioned within 1 week from iron (hydr)oxide plaques to iron sulfide plaques. During the same week, Fe(III) decreased on roots of plants not amended with sulfate but FeS did not accumulate. Prior to FeS accumulation, sulfate-amended plants had taken up the same amount of N as unamended plants. After FeS accumulation, total plant nitrogen did not increase further on sulfate-amended plants, indicating a cessation in nitrogen uptake, whereas total plant N continued to increase in unamended plants. Sulfate-amended plants produced fewer and lighter seeds with less nitrogen than unamended plants. FeS precipitation on roots may be associated with elevated sulfide and inhibited nitrogen uptake before the end of the plant’s life cycle, thus affecting the populations of this annual aquatic plant. We propose a mechanism by which a physiologically-induced decline in radial oxygen loss near the end of a plant’s life cycle initiates a precipitous decline in redox potential at the root surface and in adjacent porewater, initiating accumulation of iron sulfide plaques. These plaques could be an important locus for iron sulfide accumulation in wetland sediments.     

The role of iron in enhancing anaerobic toluene degradation in sulfate-reducing enrichment cultures

Ferrous iron enhanced the toluene degradation rate of sulfidogenic enrichment cultures inoculated with contaminated subsurface soil from an aviation fuel storage facility near the Patuxent River (Md.). Ferrous iron had an analogous effect on the degradation rate of benzoic acid, a transient metabolite of anaerobic toluene degradation in these cultures, when benzoic acid was used as a sole carbon and energy source. Two hypotheses were proposed to explain iron's effect: (a) Iron may have prevented sulfide toxicity via precipitation of sulfide as FeS, and (b) iron might have been a limiting nutrient required for degradation (i.e., amendments of iron could have compensated for iron removed from solution by precipitation as FeS). To test these hypotheses, substrate degradation rates were compared in the presence of FeSO₄ (a sulfate source that both precipitates sulfide species and precludes iron limitation) versus ZnSO₄ (a sulfate source that precipitates sulfide species but does not preclude iron limitation) versus MgSO₄ (a sulfate source that neither precipitates sulfide nor precludes iron limitation). For both toluene and benzoic acid, FeSO₄ and ZnSO₄ were comparable in their enhancement of substrate degradation rates and were superior to MgSO₄ in that respect. Thus, iron appears to ameliorate sulfide toxicity, not nutritional iron limitation, in these cultures. The observation that ethylenediaminetetraacetic acid, a chelating agent capable of retaining iron in solution in the presence of sulfide, did not stimulate the cultures is consistent with this conclusion. The implications of these results for bioremediation of fuel-contaminated aquifers that contain sulfate-reducing bacteria are discussed. Correspondence to: H.R. Beller.     

Chemical and biological mobilization of Fe(III) in marsh sediments

Iron reduction in marine sulfitic environments may occur via a mechanism involving direct bacterial reduction with the use of hydrogen as an electron donor, direct bacterial reduction involving carbon turnover, or by indirect reduction where sulfide acts to reduce iron. In the presented experiments, the relative importance of direct and indirect mechanisms of iron reduction, and the contribution of these two mechanisms to overall carbon turnover has been evaluated in two marsh environments. Sediments collected from two Northeastern US salt marshes each having different Fe (III) histories were incubated with the addition of reactive iron (as amorphous oxyhydroxide). These sediments were either incubated alone or in conjunction with sodium molybdate. Production of both inorganic and organic pore water constituents and a calculation of net carbon production were used as measures to compare the relative importance of direct bacterial reduction and indirect bacterial reduction. Results indicate that in the environments tested, the majority of the reduced iron found results from indirect reduction mediated by hydrogen sulfide, a result of dissolution and precipitation phenomena, or is a result of direct bacterial reduction using hydrogen as an electron donor. Direct iron reduction plays a minor role in carbon turnover in these environments.     

Mathematical modeling of precipitation and dissolution reactions in microbiological systems

We expand the biogeochemical model CCBATCH to include a precipitation/dissolution sub-model that contains kinetic and equilibrium options. This advancement extends CCBATCH's usefulness to situations in which microbial reactions cause or are affected by formation or dissolution of a solid phase. The kinetic option employs a rate expression that explicitly includes the intrinsic kinetics for reaction ormass-transport control, the differencefrom thermodynamic equilibrium, and the aqueous concentration of the rate-limiting metal or ligand. The equilibrium feature can be used alone, and it also serves as check that the kinetic rate never is too fast and ``overshoots' equilibrium. The features of the expanded CCBATCH are illustrated by an example in which the precipitation of Fe(OH)_{3 (s)} allows the biodegradation of citric acid, even though complexes are strong and not bioavailable. Precipitation releases citrate ligand, and biodegradation of the citrate increases the pH.     

Biogeochemistry of an Iron-Rich Hypersaline Microbial Mat (Camargue, France)

In situ microsensor measurements were combined with biogeochemical methods to determine oxygen, sulfur, and carbon cycling in microbial mats growing in a solar saltern (Salin-de-Giraud, France). Sulfate reduction rates closely followed the daily temperature changes and were highest during the day at 25°C and lowest during the night at 11°C, most probably fueled by direct substrate interactions between cyanobacteria and sulfate-reducing bacteria. Sulfate reduction was the major mineralization process during the night and the contribution of aerobic respiration to nighttime DIC production decreased. This decrease of aerobic respiration led to an increasing contribution of sulfide (and iron) oxidation to nighttime O₂ consumption. A peak of elemental sulfur in a layer of high sulfate reduction at low sulfide concentration underneath the oxic zone indicated anoxygenic photosynthesis and/or sulfide oxidation by iron, which strongly contributed to sulfide consumption. We found a significant internal carbon cycling in the mat, and sulfate reduction directly supplied DIC for photosynthesis. The mats were characterized by a high iron content of 56 mol Fe cm^–3, and iron cycling strongly controlled the sulfur cycle in the mat. This included sulfide precipitation resulting in high FeS contents with depth, and reactions of iron oxides with sulfide, especially after sunset, leading to a pronounced gap between oxygen and sulfide gradients and an unusual persistence of a pH peak in the uppermost mat layer until midnight.     

SEQUENCE Visualization of Natural Attenuation Trends at Hill Air Force Base, Utah

For monitored natural attenuation to be considered as an acceptable remedial approach, the proponent must clearly document converging lines of evidence that illustrate the effectiveness of this measure. SEQUENCE, a visualization tool based on a modified radial diagram approach, is ideally suited for evaluating spatial and temporal trends that provide supporting evidence for the efficacy of monitored natural attenuation. SEQUENCE was applied to evaluate the natural attenuation of benzene, toluene, ethylbenzene, and total xylene (BTEX) concentrations observed in groundwater at Hill Air Force Base, Utah. SEQUENCE-BTEX maps provided an efficient means of documenting the declining BTEX concentrations downgradient from the source area. SE-QUENCE-Redox maps were used to facilitate a correlation between elevated BTEX concentrations; decreasing electron acceptor concentrations (oxygen, nitrate, and sulfate); and elevated metabolic byproduct concentrations (iron(II) and methane) providing a second line of evidence that suggests BTEX compounds are being destroyed through biodegradation processes downgradient from the source area. Site-specific guidelines for interpolating representative data sets for use with the SEQUENCE approach are discussed.     

Formation of Covellite (CuS) Under Biological Sulfate-Reducing Conditions

Sulfate-reducing bacteria (SRB) play a major role in the precipitation of metal sulfides in the environment. In this work, biogenic copper sulfide formation was examined in cultures of SRB and compared to chemically initiated Cu sulfide precipitation as a reference system. Mixed cultures of SRB were incubated at 22, 45, and 60°C in nutrient solutions that contained copper sulfate. Abiotic reference samples were produced by reacting uninoculated liquid media with Na₂S solutions under otherwise identical conditions. Precipitates were collected anaerobically by centrifugation, frozen in liquid N₂, and freeze-dried, followed by analysis using X-ray diffraction (XRD), X-ray fluorescence, and scanning electron microscopy. Covellite (CuS) was the only mineral found in the precipitates. Covellite was less crystalline in the biogenic precipitates than in the abiotic samples based on XRD peak widths and peak to background ratios. Poor crystallinity may be the result of slower precipitation rates in bacterial cultures as compared to the abiotic reference systems. Furthermore, bacterial cells may inhibit the nucleation steps that lead to crystal formation. Incubation at elevated temperatures improved the crystallinity of the biotic specimens.     

Ammonia Formation By The Reduction Of Nitrite/Nitrate By Fes: Ammonia Formation Under Acidic Conditions

One issue for the origin of life under a non-reducing atmosphere is the availability of the reduced nitrogen necessary for amino acids, nucleic acids, etc. One possible source of this nitrogen is the formation of ammonia from the reduction of nitrates and nitrites produced by the shock heating of the atmosphere and subsequent chemistry. Ferrous ions will reduce these species to ammonium, but not under acidic conditions. We wish to report results on the reduction of nitrite and nitrate by another source of iron (II), ferrous sulfide, FeS. FeS reduces nitrite to ammonia at lower pHs than the corresponding reduction by aqueous Fe^{+ 2}. The reduction follows a first order decay, in nitrite concentration, with a half-life of about 150 min (room temperature, CO₂, pH 6.25). The highest product yield of ammonia measured was 53%. Under CO₂, the product yield decreases from pH 5.0 to pH 6.9. The increasing concentration of bicarbonate, at higher pH, interferes with the reaction. Comparing experiments under N₂ CO₂ shows the interference of bicarbonate. The reaction proceeds well in the presence of such species as chloride, sulfate, and phosphate, though the yield drops significantly with phosphate. FeS also reduces nitrate and, unlike with Fe^{+ 2}, the reduction shows more reproducibility. Again, the product yield decreases with increasing pH, from 7% at pH 4.7 to 0% at pH 6.9. It appears that nitrate is much more sensitive to the presence of added species, perhaps not competing as well for binding sites on the FeS surface. This may be the cause of the lack of reproducibility of nitrate reduction by Fe^{+ 2} (which also can be sensitive to binding by certain species)     

Polymersomes Containing Iron Sulfide (FeS) as Primordial Cell Model

According to Wächtershäuser??s ??Iron-Sulfur-World?? one major requirement for the development of life on the prebiotic Earth is compartmentalization. Vesicles spontaneously formed from amphiphilic components containing a specific set of molecules including sulfide minerals may have lead to the first autotrophic prebiotic units. The iron sulfide minerals may have been formed by geological conversions in the environment of deep-sea volcanos (black smokers), which can be observed even today. Wächtershäuser postulated the evolution of chemical pathways as fundamentals of the origin of life on earth. In contrast to the classical Miller-Urey experiment, depending on external energy sources, the ??Iron-Sulfur-World?? is based on the catalytic and energy reproducing redox system $ FeS + {H_2}S \to FeS{}_2 + {H_2} $ . The energy release out of this redox reaction (?_RG°?=??38 kJ/mol, pH 0) could be the cause for the subsequent synthesis of complex organic molecules and the precondition for the development of more complex units similar to cells known today. Here we show the possibility for precipitating iron sulfide inside vesicles composed of amphiphilic block-copolymers as a model system for a first prebiotic unit. Our findings could be an indication for a chemoautotrophic FeS based origin of life.     

Isolation of Thiomonas thermosulfatus Strain 51, a Species Capable of Coupling Biogenic Pyritization with Chemiosmotic Energy Transduction

The precipitation of iron sulfides potentially offers enough energy and reducing power to sustain life but organisms harnessing this reaction have not to our knowledge been previously described. We isolated a bacterial strain, capable of forming the iron sulfide minerals troilite (FeS), greigite (Fe₃S₄), and pyrite (FeS₂), from subsurface, microbial mats in Mangalia, Romania. This strain, most closely related to strains of Thiomonas sp., forms pyrite only if the redox conditions remain negative (< ?60 mV), sulfides are provided continually (≈1 mM), and the concentration of iron remains low (≤ 0.08 mM) but constant. Pyrite formation by this microbial strain is proposed as an example of biologically controlled mineralization because it is controlled by uncouplers of oxidative phosphorylation, it is larger in living than in dead cells, it is additive (controlled less by the amount of cell surfaces and more by reagents), and it results in the formation of ATP. This study indicates that precipitation and crystal formation can represent an energy resource for life and provides support for the “iron-sulfide world hypothesis” regarding the early evolution of life on Earth.     

Kinetic study of sulfide leaching by galvanic interaction between chalcopyrite, pyrite, and sphalerite in the presence of T. ferrooxidans (30 degrees C) and a thermophilic microorganism (55 degrees C)

A systematic, kinetic study and comparison of the leaching of mixed metal sulfides by galvanic conversion and in the presence of bacteria has been carried out for the first time using both powder (-100 to -400 mesh) and larger (bulk) specimen systems. The rates of dissolution of copper from chalcopyrite and zinc from sphalerite as single, electrically isolated (separate) systems were compared with electrically contacting (galvanically coupled) systems involving CuFeS(2)/FeS(2) and ZnS/FeS(2), with and without bacteria and at temperatures of 30 and 55 degrees C. The dissolution of Cu was observed to increase by a factor of 4.6 when the galvanic leaching of CuFeS(2)/FeS(2) was compared to CuFeS(2) leaching at 30 degrees C. When bacteria were present, Cu dissolution increased by an additional factor of 2.1 in the CuFeS(2)/FeS(2) system. At 55 degrees C, the corresponding ratios for Cu were 4.3 and 2.7, respectively. The galvanic leaching of Zn in the ZnS/FeS(2) system compared to ZnS leaching increased by a factor of 2 at 30 degrees C; in the presence of bacteria the dissolution of Zn from the ZnS/FeS(2) system increased by an additional factor of 1.3 at the same temperature. By comparison, the ratio of Cu dissolution from CuFeS(2) in acid-bacterial medium and sterile controls (without bacteria) was 5.5. The corresponding ratio for Zn from ZnS was 2.2 at both 30 and 55 degrees C. The order of reaction was found to be essentially first order for the leaching of powder systems at both 30 and 55 degrees C (with T. Ferrooxidans and thermophilic microorganisms, respectively). The corresponding reaction rate constants were observed to be 12.6 and 22.9 for T. ferrooxidans and the thermophilic microorganisms, respectively. Activation energies for the various systems were also determined.     

Thermophilic biodegradation of BTEX by two Thermus Species

Two thermophilic bacteria, Thermus aquaticus ATCC 25104 and Thermus species ATCC 27978, were investigated for their abilities to degrade BTEX (benzene, toluene, ethylbenzene, and xylenes). Thermus aquaticus and the Thermus sp. were grown in a nominal medium at 70 degrees C and 60 degrees C, respectively, and resting cell suspensions were used to study BTEX biodegradation at the same corresponding temperatures. The degradation of BTEX by these cell suspensions was measured in sealed serum bottles against controls that also displayed significant abiotic removals of BTEX under such high-temperature conditions. For T. aquaticus at a suspension density of only 1.3 x 10(7) cells/mL and an aqueous total BTEX concentration of 2.04 mg/L (0.022 mM), benzene, toluene, ethylbenzene, m-xylene, and an unresolved mixture of o-and p-xylenes were biodegraded by 10, 12, 18, 20, and 20%, respectively, after 45 days of incubation at 70 degrees C. For the Thermus sp. at a suspension density of 1.1 x 10(7) cells/mL and an aqueous total BTEX concentration of 6.98 mg/L (0.079 mM), benzene, toluene, ethylbenzene, m-xylene, and the unresolved mixture of o-and p-xylenes were biodegraded by 40, 35, 32, 33, and 33%, respectively, after 45 days of incubation at 60 degrees C. Raising the BTEX concentrations lowered the extents of biodegradation. The biodegradations of both benzene and toluene were enhanced when T. aquaticus and the Thermus sp. were pregrown on catechol and o-cresol, respectively, as carbon sources. Use of [U-(14)C]benzene and [ring-(14)C]toluene verified that a small fraction of these two compounds was metabolized within 7 days to water-soluble products and CO(2) by these nongrowing cell suspensions. Our investigation also revealed that the nominal medium can be simplified by eliminating the yeast extract and using a higher tryptone concentration (0.2%) without affecting the growth and BTEX degrading activities of these cells. (c) 1995 John Wiley & Sons, Inc.     

Aerobic MTBE biodegradation in the presence of BTEX by two consortia under batch and semi-batch conditions

This study explores the effect of microbial consortium composition and reactor configuration on methyl tert-butyl ether (MTBE) biodegradation in the presence of benzene, toluene, ethylbenzene and p-xylenes(BTEX). MTBE biodegradation was monitored in the presence and absence of BTEX in duplicate batch reactors inoculated with distinct enrichment cultures: MTBE only (MO—originally enriched on MTBE) and/or MTBE BTEX (MB—originally enriched on MTBE and BTEX). The MO culture was also applied in a semi-batch reactor which received both MTBE and BTEX periodically in fresh medium after allowing cells to settle. The composition of the microbial consortia was explored using a combination of 16S rRNA gene cloning and quantitative polymerase chain reaction targeting the known MTBE-degrading strain PM1^T. MTBE biodegradation was completely inhibited by BTEX in the batch reactors inoculated with the MB culture, and severely retarded in those inoculated with the MO culture (0.18 ± 0.04 mg/L-day). In the semi-batch reactor, however, the MTBE biodegradation rate in the presence of BTEX was almost three times as high as in the batch reactors (0.48 ± 0.2 mg/L-day), but still slower than MTBE biodegradation in the absence of BTEX in the MO-inoculated batch reactors (1.47 ± 0.47 mg/L-day). A long lag phase in MTBE biodegradation was observed in batch reactors inoculated with the MB culture (20 days), but the ultimate rate was comparable to the MO culture (0.95 ± 0.44 mg/L-day). Analysis of the cultures revealed that strain PM1^T concentrations were lower in cultures that successfully biodegraded MTBE in the presence of BTEX. Also, other MTBE degraders, such as Leptothrix sp. and Hydrogenophaga sp. were found in these cultures. These results demonstrate that MTBE bioremediation in the presence of BTEX is feasible, and that culture composition and reactor configuration are key factors.