Effect of soil organic matter on petroleum hydrocarbon degradation in diesel/fuel oil-contaminated soil.

The purpose of this study is to investigate the effect of soil organic matter (SOM) content levels on the biodegradation of total petroleum hydrocarbons (TPH). Batch experiments were conducted with soils with 2% or 10% organic matter that had been contaminated by diesel or fuel oil. In addition to the TPH (diesel or fuel oil) degradation efficiency, a comprehensive investigation was conducted on the TPH-degrading microbial community using molecular tools including oligonucleotide microarray technique and terminal restriction fragment length polymorphism analysis (T-RFLP). TPH was reduced from 10,000 mg/kg to 1849-4352 mg/kg dry weight soil. Higher biodegradation efficiencies and kinetic rate constants were observed in higher SOM contents. Hydrocarbon fractional analyses were conducted to explain the optimal operation with relatively low resin and aromatic fractions detected at the end of the remediation. The bacterial and fungal counts in the 10% SOM were approximately 10 CFU/g to 102 CFU/g above those in the 2% SOM, and the lowest fungal level was found when the least TPH degradability was measured. The internal transcribed spacer microarray identified the microorganisms that were introduced and proved their survival. The associated growth pattern confirmed that different kinds of contamination oils affected the microbial community diversity over time. Both the microarray and T-RFLP profiles indicated that Gordonia alkanivorans, G. desulfuricans, and Rhodococcus erythoropolis were the dominant bacteria, while Fusarium oxysporum and Aspergillus versicolor were the dominant fungi. The T-RFLP-derived nonmetric multidimensional scaling concluded that the dynamics of the microbial communities were impacted by the TPH degradation stages.

Using metabolic charge production in the tricarboxylic acid cycle (QTCA) to evaluate the extracellular-electron-transfer performances of Shewanella spp.

Using an electrochemical cell equipped with carbon felt electrodes (poised at +0.63 V vs. SHE), the current production capabilities of two Shewanella strains-NTOU1 and KR-12-were examined under various conditions with lactate as an electron donor. The metabolic charge produced in the tricarboxylic acid cycle (QTCA) was calculated by mass-balance. The data showed a linear relation between the electric coulomb production (QEL) and QTCA with an R2 of 0.65. In addition, a large amount of pyruvate accumulation was observed at pH = 6, rendering QTCA negative. The results indicate an occurrence of an undesired cataplerotic reaction. It was also found that QTCA provides important information showing the oxygen-boosting TCA cycle and anodic-current generation of Shewanella spp. Linear dependence of the change in charge for biomass growth (4.52FΔnCell) on QTCA was also found as expressed by 4.52FΔnCell = 1.0428 QTCA + 0.0442, indicating that these two charge quantities are inherently identical under most of the experimental conditions. In the mediator-spiked experiments, the external addition of the mediators (ferricyanide, anthraquinone-2, 6-disulfonate, and riboflavin) beyond certain concentrations inhibited the activity of the TCA cycle, indicating that the oxidative phosphorylation is deactivated by excessive amounts of mediators, yet Shewanella spp. are constrained with regard to carrying out the substrate-level phosphorylation.

Amylolysis is predominated by cell-surface-bound hydrolase during anaerobic fermentation under mesophilic conditions.

While knowing the amylolysis mechanism is important to effectively decompose corn starch fed into an anaerobic digestor, the objective of this study was to detect the activities and locations of α-amylase in a continuous reactor and batch cultures. In the continuous reactor operated at 35 °C, the greatest cell-bound α-amylase activity was found to be 4.7 CU mL-1 at hydraulic retention time (HRT) = 9 h, while the greatest volumetric hydrogen production rate (rH2) was observed at HRT = 3 h as 61 mmol L-1 day-1. In the batch tests, the cell-bound α-amylase activities increased when the carbohydrate concentration decreased, and no significant reducing sugar accumulation was found in the serum bottles. By examining the specific hydrogen production rate (qH2) against different corn starch concentrations, the half-saturation constant (KSta) and the maximum qH2 were regressed to be 0.47 g L-1 and 6 mmol g-VSS-1 d-1, respectively. The electronic microscopic images showed that the microbes could colonize on the starch granules without the disturbance of any floc-like materials. Conclusively, by excluding the methanogens and floc matrix, the secreted α-amylases are predominately bound on the cell surfaces and enabled the microbial cells favorably attach on large substrates for hydrolysis under the mesophilic condition.

A metabolic-activity-detecting approach to life detection: Restoring a chemostat from stop-feeding using a rapid bioactivity assay.

A mediated glassy carbon electrode covered by a thin-film polyviologen was used in the present study to rapidly detect bioactivity in a mixed-culture chemostat (dominated by Clostridium sp.). With the addition of 1mM hexacyanoferrate and 9mM glucose, the current increasing rate (dI/dt) measured under a poised potential of 500mV (vs. Ag/AgCl) can be defined as the quantity of metabolic activity. In the experiment of restoring the chemostat from stop-feeding, it is suggested that when the dI/dt was >2µAmin-1, the influent pump could be directly turned on to maintain the high dilution rate of 0.5h-1; when the dI/dt was lower than 2µAmin-1, reducing the dilution rate would be needed to avoid cell wash out. Since the soluble mediators and polyviologen film will enhance performances by favorable electron transfer and positively charged surfaces, respectively, we suggest that the method can also be employed to detect the bioactivities in environmental samples.

A combined upflow anaerobic sludge bed, aerobic, and anoxic fixed-bed reactor system for removing tetramethylammonium hydroxide and nitrogen from light-emitting diode wastewater.

A laboratory study using a combined upflow anaerobic sludge bed (UASB) and aerobic and anoxic fixed-bed reactor system was undertaken to explore its capability for removing tetramethylammonium hydroxide (TMAH) and nitrogen from light-emitting diode wastewater. When the organic loading rate was maintained at 0.26-0.65 kg TMAH m(-3 )d(-1), the UASB reactor removed 70-100% of TMAH through methanogenesis. When the [Formula: see text] -N loading rate was maintained at 0.73-1.4 kg [Formula: see text]-N m(-3 )d(-1), the aerobic reactor oxidized 31-59% of [Formula: see text]-N to [Formula: see text]-N through nitritation. When the nitrogen loading rate was maintained at 0.42-0.75 kg N m(-3 )d(-1), the anoxic reactor removed 27-63% of nitrogen through anammox. The performance data of the combined reactor system agreed well with the stoichiometric relationships of methanogenesis, nitritation, and anammox. The batch studies showed that a higher initial TMAH concentration of up to 2520 mg L(-1) gave a higher methanogenic activity of up to 16 mL CH4 g(-1) VSS d(-1). An increase in the initial TMAH concentration of up to 500 mg L(-1) gradually decreased the activity of ammonia-oxidizing bacteria; whereas an increase in the initial TMAH concentration of up to 47 mg L(-1) imposed a marked inhibiting effect on the activity of anammox bacteria.

Removal of nitrogen from secondary effluent of a petrochemical industrial park by a hybrid biofilm-carrier reactor with one-stage ANAMMOX.

A laboratory study was undertaken to explore the capability of one-stage ANAMMOX in a hybrid biofilm-carrier reactor (HBCR) fed with petrochemical wastewater. Under favorable operating conditions in continuous-flow operations (at the dissolved oxygen level of 0.5-1.0 mg L(-1)), the average total nitrogen (TN) removal efficiency reached 62-67% and approximately 90% of TN can be removed by ANAMMOX. In batch operations of the hybrid biofilm-carrier reactor (without adding carbon substrate), the specific TN removal rate of the reactor in which both Kaldnes and nonwoven carriers were kept was two-fold higher than that of the reactor in which only nonwoven carriers were kept. This indicated that the microbial activity of thinner biofilms (Kaldnes carriers) was remarkably higher than that of thicker biofilms (nonwoven carriers). Finally, based on the 16S rRNA clone library, a cluster of ANAMMOX Candidatus Kuenenia stuttgartiensis was identified.

Impact of food to microorganism (F/M) ratio and colloidal chemical oxygen demand on nitrification performance of a full-scale membrane bioreactor treating thin film transistor liquid crystal display wastewater.

This study investigated impact of food to microorganism (F/M) ratio and colloidal chemical oxygen demand (COD) on nitrification performance in one full-scale membrane bioreactor (MBR) treating monoethanolamine (MEA)/dimethyl sulfoxide (DMSO)-containing thin film transistor liquid crystal display (TFT-LCD) wastewater. Poor nitrification was observed under high organic loading and high colloidal COD conditions, suggesting that high F/M ratio and colloidal COD situations should be avoided to minimize their negative impacts on nitrification. According to the nonmetric multidimensional scaling (NMS) statistical analyses on terminal restriction fragment length polymorphism (T-RFLP) results of ammonia monooxygenase (amoA) gene, the occurrence of Nitrosomonas oligotropha-like ammonia oxidizing bacteria (AOB) was positively related to successful nitrification in the MBR systems, while Nitrosomonas europaea-like AOB was positively linked to nitrification rate, which can be attributed to the high influent total nitrogen condition. Furthermore, Nitrobacter- and Nitrospira-like nitrite oxidizing bacteria (NOB) were both abundant in the MBR systems, but the continuously low nitrite environment is likely to promote the growth of Nitrospira-like NOB.

WITHDRAWN: Bioaugmentation strategies to improve cellulolytic and hydrogen-producing characteristics of CSTR intermittent fed with vegetable kitchen waste and napiergrass.

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Nitrification performance and microbial ecology of nitrifying bacteria in a full-scale membrane bioreactor treating TFT-LCD wastewater.

This study investigated nitrification performance and nitrifying community in one full-scale membrane bioreactor (MBR) treating TFT-LCD wastewater. For the A/O MBR system treating monoethanolamine (MEA) and dimethyl sulfoxide (DMSO), no nitrification was observed, due presumably to high organic loading, high colloidal COD, low DO, and low hydraulic retention time (HRT) conditions. By including additional A/O or O/A tanks, the A/O/A/O MBR and the O/A/O MBR were able to perform successful nitrification. The real-time PCR results for quantification of nitrifying populations showed a high correlation to nitrification performance, and can be a good indicator of stable nitrification. Terminal restriction fragment length polymorphism (T-RFLP) results of functional gene, amoA, suggest that Nitrosomonas oligotropha-like AOB seemed to be important to a good nitrification in the MBR system. In the MBR system, Nitrobacter- and Nitrospira-like NOB were both abundant, but the low nitrite environment is likely to promote the growth of Nitrospira-like NOB.

Evaluation of the optimal strategy for ex situ bioremediation of diesel oil-contaminated soil.

PURPOSE: Bioaugmentation and biostimulation have been widely applied in the remediation of oil contamination. However, ambiguous results have been reported. It is important to reveal the controlling factors on the field for optimal selection of remediation strategy. In this study, an integrated field landfarming technique was carried out to assess the relative effectiveness of five biological approaches on diesel degradation. The limiting factors during the degradation process were discussed. METHOD: A total of five treatments were tested, including conventional landfarming, nutrient enhancement (NE), biosurfactant addition (BS), bioaugmentation (BA), and combination of bioaugmentation and biosurfactant addition (BAS). The consortium consisted of four diesel-degrading bacteria strains. Rhamnolipid was used as the biosurfactant. The diesel concentration, bacterial population, evolution of CO(2), and bacterial community in the soil were periodically measured. RESULTS: The best overall degradation efficiency was achieved by BAS treatment (90 ± 2%), followed by BA (86 ± 2%), NE (84 ± 3%), BS (78 ± 3%), and conventional landfarming (68 ± 3%). In the early stage, the total petroleum hydrocarbon was degraded 10 times faster than the degradation rates measured during the period from day 30 to 100. At the later stage, the degradation rates were similar among treatments. In the conventional landfarming, contaminated soil contained bacteria ready for diesel degradation. CONCLUSION: The availability of hydrocarbon was likely the limiting factor in the beginning of the degradation process. At the later stage, the degradation was likely limited by desorption and mass transfer of hydrocarbon in the soil matrix.

Strategy of controlling the volumetric loading rate to promote hydrogen-production performance in a mesophilic-kitchen-waste fermentor and the microbial ecology analyses.

The kitchen waste was chosen as a high solid (42 gL(-1) of volatile suspended solid, VSS) and high organic (107 gL(-1) of chemical oxygen demand) feedstock for operating a 3-L mesophilic fermentor. The greatest specific hydrogen production rate ( r(H2) was observed in Stage 3 as 3.4 L-H2 L(-1) day(-1) with a volumetric loading rate (VLR) of 100 g-CODL(-1) day(-1); the highest hydrogen yield was observed in Stage 2 as 96 mL-H2 g(-1) of influent VSS with a VLR of 46 g-COD L(-1) day(-1). In Stages 1 (with a VLR of 27 g-COD L(-1)) and 2, the sum of Butyrivibrio fibrisolvens and Clostridium proteoclasticum is dominant, but in Stage 3, Olsenella genomosp, became dominant and constituted 44% of the entire population. The dependence of VLR and r(H2)could be regressed as a linear equation of r(H2) = (2.83 VLR + 40.5) x 10(-2) .

Effects of oxygen on Shewanella decolorationis NTOU1 electron transfer to carbon-felt electrodes.

To clarify the major factor caused by oxygen-enhancing charge production of Shewanella decolorationis NTOU1 towards a polarized anode, a series of experimental runs (i.e., with/without ambient air flushing and with/without ammonia addition as nitrogen source) were conducted in this study. Within 6-day of operation at +0.4 V vs. Ag|AgCl and starting with 35 mM of lactate, consistently the electrical charge production under the aerobic condition was higher than that under the anaerobic condition. In all the experimental runs, the values of nicotinamide adenine dinucleotide (NADH) production were found to be correlated positively and significantly with the charge production, but the highest Coulombic efficiency of 18% was observed under the anaerobic conditions without ammonia addition while the lowest charge production occurred. Those results indicate that NADH production enhanced by oxygen is the leading cause of the increase of the charge production, but the biomass production and the oxygen reduction would both consume NADH electrons and lead to lower electron recoveries. In addition, whether under constant aerobic or anaerobic, or alternating aerobic/anaerobic conditions, chronoamperometric results made it possible to rule out other factors, like lactate uptake rate or cell growth, which might increase the charge production under aerobic conditions. By using high performance liquid chromatography, some diffusive flavins (e.g., 0.5 microM of riboflavin) were found under the aerobic condition, but were not found under the anaerobic one. However, from results of cyclic voltammetry (CV), the signals of flavins were found to be approximately the same under both conditions. Although it is inferred that oxygen renders the flavins secreted extracellularly, that is not the major effect of oxygen for boosting the charge production. Furthermore, bound flavins under anaerobic condition were found to be effectively electrocatalytic according to sigmoidal CV result.

Ex situ bioremediation of oil-contaminated soil.

An innovative bioprocess method, Systematic Environmental Molecular Bioremediation Technology (SEMBT) that combines bioaugmentation and biostimulation with a molecular monitoring microarray biochip, was developed as an integrated bioremediation technology to treat S- and T-series biopiles by using the landfarming operation and reseeding process to enhance the bioremediation efficiency. After 28 days of the bioremediation process, diesel oil (TPH(C10-C28)) and fuel oil (TPH(C10-C40)) were degraded up to approximately 70% and 63% respectively in the S-series biopiles. When the bioaugmentation and biostimulation were applied in the beginning of bioremediation, the microbial concentration increased from approximately 10(5) to 10(6) CFU/g dry soil along with the TPH biodegradation. Analysis of microbial diversity in the contaminated soils by microarray biochips revealed that Acinetobacter sp. and Pseudomonas aeruginosa were the predominant groups in indigenous consortia, while the augmented consortia were Gordonia alkanivorans and Rhodococcus erythropolis in both series of biopiles during bioremediation. Microbial respiration as influenced by the microbial activity reflected directly the active microbial population and indirectly the biodegradation of TPH. Field experimental results showed that the residual TPH concentration in the complex biopile was reduced to less than 500 mg TPH/kg dry soil. The above results demonstrated that the SEMBT technology is a feasible alternative to bioremediate the oil-contaminated soil.

Application of rhamnolipid and surfactin for enhanced diesel biodegradation--effects of pH and ammonium addition.

This study investigated the effects of pH and ammonium concentrations on the potential application of two biosurfactants, surfactin (SF) and rhamnolipid (RL), for enhanced diesel biodegradation with a series of bench-scale experiments. In general, compared to the experiments without biosurfactant addition, adding RL or SF to diesel-water systems at concentrations above their critical micelle concentration (CMC) values benefited diesel emulsification, and therefore enhanced diesel biodegradation. The effects of pH on RL or SF-enhanced biodegradation of diesel were in good agreement with the trends of emulsion index values for RL or SF addition, respectively, under different pH conditions, suggesting that enhanced diesel emulsification by RL or SF addition promoted biodegradation of diesel. In diesel-water systems with 50mg/L of RL addition, an optimum pH condition for microbial growth and diesel biodegradation was found to be at a pH 7.2, while decreasing pH to 5.2 or increasing it to 8.4 reduced those parameters considerably. For the cases where 40 mg/L of SF was added, the enhancing ability shared a general trend with that observed for adding 50mg/L of RL as the pH increased from 5.2 to 7.2. Further increase of pH to 8.4, however, did not seem to negatively influence biodegradation and biomass growth. With respect to the effects of ammonium concentration on diesel biodegradation in diesel-water systems with 50mg/L of RL addition, an optimum ammonium addition for microbial growth and diesel biodegradation was found between 200 and 300 mg-N/L, but a dramatic decrease in growth and biodegradation occurred at ammonium addition up to 450 mg-N/L. For the cases where 40 mg/L of SF was added, an increase of ammonium addition from 50 to 200mg-N/L substantially increased microbial growth and biodegradation of diesel. Further increase of ammonium concentration to 450 mg-N/L, however, did not further improve diesel biodegradation.

Application of biosurfactants, rhamnolipid, and surfactin, for enhanced biodegradation of diesel-contaminated water and soil.

This study investigated potential application of two biosurfactants, surfactin (SF) and rhamnolipid (RL), for enhanced biodegradation of diesel-contaminated water and soil with a series of bench-scale experiments. The rhamnolipid used in this study, a commonly isolated glycolipid biosurfactant, was produced by Pseudomonas aeruginosa J4, while the surfactin, a lipoprotein type biosurfactant, was produced by Bacillus subtilis ATCC 21332. Both biosurfactants were able to reduce surface tension to less than 30 dynes/cm from 72 dynes/cm with critical micelle concentration (CMC) values of 45 and 50 mg/L for surfactin and rhamnolipid, respectively. In addition, the results of diesel dissolution experiments also demonstrated their ability in increasing diesel solubility with increased biosurfactant addition. In diesel/water batch experiments, an addition of 40 mg/L of surfactin significantly enhanced biomass growth (2500 mg VSS/L) as well as increased diesel biodegradation percentage (94%), compared to batch experiments with no surfactin addition (1000 mg VSS/L and 40% biodegradation percentage). Addition of surfactin more than 40 mg/L, however, decreased both biomass growth and diesel biodegradation efficiency, with a worse diesel biodegradation percentage (0%) at 400 mg/L of SF addition. Similar trends were also observed for both specific rate constants of biomass growth and diesel degradation, as surfactin addition increased from 0 to 400 mg/L. Addition of rhamnolipid to diesel/water systems from 0 to 80 mg/L substantially increased biomass growth and diesel biodegradation percentage from 1000 to 2500 mg VSS/L and 40 to 100%, respectively. Rhamnolipid addition at a concentration of 160 mg/L provided similar results to those of an 80 mg/L addition. Finally, potential application of surfactin and rhamnolipid in stimulating indigenous microorganisms for enhanced bioremediation of diesel-contaminated soil was also examined. The results confirmed their enhancing capability on both efficiency and rate of diesel biodegradation in diesel/soil systems.

Self-excreted mediator from Escherichia coli K-12 for electron transfer to carbon electrodes.

Escherichia coli K-12 was cultured under anaerobic conditions to form biofilm on carbon fiber electrodes in glucose-containing medium. The anodic current increased with the development of the biofilm and depended on the glucose concentration. Cyclic voltammetric results support the presence of a redox compound(s) excreted from E. coli cells in the biofilm. The compound remained in the film under conditions of continuous flow and gave a couple of oxidation and reduction waves, which may be assigned to a menaquinone-like compound based on the mid-point potential (-0.22 V vs Ag|AgCl at pH 7.1) and its pH dependence. The catalytic current started to increase around the anodic peak potential of the redox compound and also increased by the permeabilization of the E. coli cell membranes with ethylenediamine tetraacetic acid-treatment. The results indicate that the E. coli-excreted redox compound works as a mediator for the electron transfer from the E. coli cells to the electrode as the final electron acceptor. The activity of the redox compound in the E. coli-biofilm as a mediator with some mobility was also verified for diaphorase-catalyzed electrochemical oxidation of NADH.

A dual-substrate steady-state model for biological hydrogen production in an anaerobic hydrogen fermentation process.

Biological hydrogen production from anaerobic waste fermentation possesses potential benefits in simultaneously reducing organic wastes and generating sustainable energy sources. Three kinetic-based steady-state models for anaerobic fermentation of multiple substrates, including glucose and peptone, were evaluated. Experimental results obtained from a continuous stirred tank reactor (CSTR) were primarily used for model evaluation. The dual-substrate steady-state model developed and the associated kinetic parameters estimated in this study successfully described the anaerobic growth of hydrogen-producing bacteria. The model was able to capture the general trends of consumption of substrates and accumulation of products, including formate, acetate, butyrate, and hydrogen, at dilution rates (D) between 0.06 and 0.69/h. According to the model, the adverse effects of endogeneous and peptone metabolism on net hydrogen production can be minimized by increasing D. For the operational conditions of D > 0.69/h, however, substantial washout of hydrogen-producing bacteria from the CSTR was observed, and it resulted in a rapid drop in hydrogen production rate as well.

Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens.

An anaerobic phthalate isomer-degrading strain (JT(T)) that we previously isolated was characterized. In addition, a strictly anaerobic, mesophilic, syntrophic phthalate isomer-degrading bacterium, designated strain JI(T), was isolated and characterized in this study. Both were non-motile rods that formed spores. In both strains, the optimal growth was observed at temperatures around 37 degrees C and neutral pH. In syntrophic co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei, both strains could utilize two or three phthalate isomers for growth, and produce acetate and methane as end products. Strain JT(T) was able to grow on isophthalate, terephthalate, and a number of low-molecular weight aromatic compounds, such as benzoate, hydroquinone, 2-hydroxybenzoate, 3-hydroxybenzoate, 2,5-dihydroxybenzoate, 3-phenylpropionate in co-culture with M. hungatei. It could also grow on crotonate, hydroquinone and 2,5-dihydroxybenzoate in pure culture. Strain JI(T) utilized all of the three phthalate isomers as well as benzoate and 3-hydroxybenzoate for growth in co-culture with M. hungatei. No substrates were, however, found to support the axenic growth of strain JI(T). Neither strain JT(T) nor strain JI(T) could utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, Fe (III) or 4-hydroxybenzoate as electron acceptor. Phylogenetically, strains JT(T) and JI(T) were relatively close to the members of the genera Pelotomaculum and Cryptanaerobacter in 'Desulfotomaculum lineage I'. Physiological and chemotaxonomic characteristics indicated that the two isolates should be classified into the genus Pelotomaculum, creating two novel species for them. Here, we propose Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov. for strain JT(T) and strain JI(T), respectively. The type strains are strains JT(T) (= DSM 16121(T )= JCM 11824(T )= NBRC 100523(T)) and JI(T) (= JCM 12282(T) = BAA-1053(T)) for P. terephthalicum and P. isophthalicum, respectively.

Escherichia coli-catalyzed bioelectrochemical oxidation of acetate in the presence of mediators.

Bioelectrocatalytic oxidation of acetate was investigated under anaerobic conditions by using Escherichia coli K-12 (IFO 3301) cells cultured on aerobic media containing poly-peptone, glucose or acetate as the sole carbon source. It was found that all E. coli cells cultured on the three media work as good catalysts of the electrochemical oxidation of acetate as well as glucose with Fe(CN)6(3-), 2,3-dimethoxy-5-methyl-1,4-benzo-quinone (Q0), 2,6-dichloro-indophenol, or 2-methyl-1,4-naphthoquinone as artificial electron acceptors (mediators). Acetate-grown E. coli cells exhibited the highest relative activity of the acetate oxidation against the glucose oxidation. On the other hand, all the artificial electron acceptors used work as inhibitors for the catalytic oxidation of acetate at increased concentrations. The inhibition phenomenon can be interpreted in terms of competitive substrate inhibition as a whole. Apparent values of Michaelis constant, catalytic constant, and inhibition constant were evaluated by amperometric methods. Q0 is an effective artificial mediator as evidenced by a large reaction rate constant between the cell and Q0 at least at low concentrations (<50 microM). However, Fe(CN)6(3-) is a promising mediator in biosensor applications because the inhibition constant is very large and it works as an electron acceptor even under aerobic conditions.

Identification and isolation of anaerobic, syntrophic phthalate isomer-degrading microbes from methanogenic sludges treating wastewater from terephthalate manufacturing.

The microbial populations responsible for anaerobic degradation of phthalate isomers were investigated by enrichment and isolation of those microbes from anaerobic sludge treating wastewater from the manufacturing of terephthalic acid. Primary enrichments were made with each of three phthalate isomers (ortho-, iso-, and terephthalate) as the sole energy source at 37 degrees C with two sources of anaerobic sludge (both had been used to treat wastewater containing high concentrations of phthalate isomers) as the inoculum. Six methanogenic enrichment cultures were obtained which not only degraded the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with concomitant production of methane, presumably involving strictly syntrophic substrate degradation. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant bacteria in the enrichment cultures were affiliated with a recently recognized non-sulfate-reducing subcluster (subcluster Ih) in the group 'Desulfotomaculum lineage I' or a clone cluster (group TA) in the class delta-PROTEOBACTERIA: Several attempts were made to isolate these microbes, resulting in the isolation of a terephthalate-degrading bacterium, designated strain JT, in pure culture. A coculture of the strain with the hydrogenotrophic methanogen Methanospirillum hungatei converted terephthalate to acetate and methane within 3 months of incubation, whereas strain JT could not degrade terephthalate in pure culture. During the degradation of terephthalate, a small amount of benzoate was transiently accumulated as an intermediate, indicative of decarboxylation of terephthalate to benzoate as the initial step of the degradation. 16S rRNA gene sequence analysis revealed that the strain was a member of subcluster Ih of the group 'Desulfotomaculum lineage I', but it was only distantly related to other known species.