Biodegradation Of Benzoate Research Articles

Zero-valent iron-induced successive chemical transformation and biodegradation of lindane in historically contaminated soil: An isotope-informed metagenomic study

Zero-valent iron (ZVI) is widely used to mitigate environmental pollutants such as chlorinated pesticides through reductive reactions accompanied by extensive impacts on the soil microbial community. However, whether and how ZVI changes the biodegradation of target compounds remain poorly understood. Here, we monitor the fate of lindane using a 14C-labled tracer and evaluate the growth and functions of the bacterial community in ZVI-stressed conditions in a historically γ-hexachlorocyclohexane (lindane)-contaminated soil using a combination of isotopic (18O-H2O) and metagenomic methods. ZVI promoted the biomineralization of lindane in a dose-dependent manner. Soil bacteria were inhibited by amendment with ZVI during the initial stages of incubation (first three days) but recovered during the subsequent six weeks. Metagenomic study indicates that the todC1/bedC1 genes involved in the oxidation of dechlorinated lindane intermediates were upregulated in the 18O-labeled bacterial community but the presence of the lin genes responsible for lindane dechlorination was not confirmed. In addition, the benzoate biodegradation pathway that links to downstream catabolism of lindane was enhanced. These findings indicate successive chemical and biological degradation mechanisms underlying ZVI-enhanced lindane mineralization and provide a scientific basis for the inclusion of an extended bioremediation stage in the environmental application of ZVI materials.

Predicted functional genes for the biodegradation of xenobiotics in groundwater and sediment at two contaminated naval sites.

The goals of this study were to predict the genes associated with the biodegradation of organic contaminants and to examine microbial community structure in samples from two contaminated sites. The approach involved a predictive bioinformatics tool (PICRUSt2) targeting genes from twelve KEGG xenobiotic biodegradation pathways (benzoate, chloroalkane and chloroalkene, chlorocyclohexane and chlorobenzene, toluene, xylene, nitrotoluene, ethylbenzene, styrene, dioxin, naphthalene, polycyclic aromatic hydrocarbons, and metabolism of xenobiotics by cytochrome P450). Further, the predicted phylotypes associated with functional genes early in each pathway were determined. Phylogenetic analysis indicated a greater diversity in the sediment compared to the groundwater samples. The most abundant genera for sediments/microcosms included Pseudomonas, Methylotenera, Rhodococcus, Stenotrophomonas, and Brevundimonas, and the most abundant for the groundwater/microcosms included Pseudomonas, Cupriavidus, Azospira, Rhodococcus, and unclassified Burkholderiaceae. Genes from all twelve of the KEGG pathways were predicted to occur. Seven pathways contained less than twenty-five genes. The predicted genes were lowest for xenobiotics metabolism by cytochrome P450 and ethylbenzene biodegradation and highest for benzoate biodegradation. Notable trends include the occurrence of the first genes for trinitrotoluene and 2,4-dinitrotoluene degradation. Also, the complete path from toluene to benzoyl-CoA was predicted. Twenty-two of the dioxin pathway genes were predicted, including genes within the first steps. The following phylotypes were associated with the greatest number of pathways: unclassified Burkholderiaceae, Burkholderia-Caballeronia-Paraburkholderia, Pseudomonas, Rhodococcus, unclassified Betaproteobacteria, and Polaromonas. This work illustrates the value of PICRUSt2 for predicting biodegradation potential and suggests that a subset of phylotypes could be important for the breakdown of organic contaminants or their metabolites. KEY POINTS: • The approach is a low-cost alternative to shotgun sequencing. • The genes and phylotypes encoding for xenobiotic degradation were predicted. • A subset of phylotypes were associated with many pathways.

Integrated Microbiome-Metabolome Analysis Reveals Stage-Dependent Alterations in Bacterial Degradation of Aromatics in Leptinotarsa decemlineata.

To avoid potential harm during pupation, the Colorado potato beetle Leptinotarsa decemlineata lives in two different habitats throughout its developmental excursion, with the larva and adult settling on potato plants and the pupa in soil. Potato plants and agricultural soil contain a specific subset of aromatics. In the present study, we intended to determine whether the stage-specific bacterial flora plays a role in the catabolism of aromatics in L. decemlineata. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the operational taxonomic units (OTUs) obtained by sequencing of culture-independent 16S rRNA region enriched a group of bacterial genes involved in the elimination of mono- and polycyclic aromatics at the pupal stage compared with those at the larval and adult periods. Consistently, metabolome analysis revealed that dozens of monoaromatics such as styrene, benzoates, and phenols, polycyclic aromatics, for instance, naphthalene and steroids, were more abundant in the pupal sample. Moreover, a total of seven active pathways were uncovered in the pupal specimen. These ways were associated with the biodegradation of benzoate, 4-methoxybenzoate, fluorobenzoates, styrene, vanillin, benzamide, and naphthalene. In addition, the metabolomic profiles and the catabolism abilities were significantly different in the pupae where their bacteria were removed by a mixture of three antibiotics. Therefore, our data suggested the stage-dependent alterations in bacterial breakdown of aromatics in L. decemlineata.

Uncovering Competitive and Restorative Effects of Macro- and Micronutrients on Sodium Benzoate Biodegradation.

A model aromatic compound, sodium benzoate, is generally used for simulating aromatic pollutants present in textile effluents. Bioremediation of sodium benzoate was studied using the most abundant bacteria, Pseudomonas citronellolis, isolated from the effluent treatment plants of South Gujarat, India. Multiple nutrients constituting the effluent in actual conditions are proposed to have interactive effects on biodegradation which needs to be analyzed strategically for successful field application of developed bioremediation process. Two explicitly different sets of fractional factorial designs were used to investigate the interactive influence of alternative carbon, nitrogen sources, and inorganic micronutrients on sodium benzoate degradation. The process was negatively influenced by the co-existence of other carbon sources and higher concentration of KH2PO4 whereas NH4Cl and MgSO4 exhibited positive effects. Optimized concentrations of NH4Cl, MgSO4, and KH2PO4 were found to be 0.35, 1.056, and 0.3 mg L–1 respectively by central composite designing. The negative effect of high amount of KH2PO4 could be ameliorated by increasing the amount of NH4Cl in the biodegradation milieu indicating the possibility of restoration of the degradation capability for sodium benzoate degradation in the presence of higher phosphate concentration.

Enhanced Anaerobic Biodegradation of Benzoate Under Sulfate-Reducing Conditions With Conductive Iron-Oxides in Sediment of Pearl River Estuary.

Anaerobic biodegradation of aromatic compounds under sulfate-reducing conditions is important to marine sediments. Sulfate respiration by a single bacterial strain and syntrophic metabolism by a syntrophic bacterial consortium are primary strategies for sulfate-dependent biodegradation of aromatic compounds. The objective of this study was to investigate the potential of conductive iron oxides to facilitate the degradation of aromatic compounds under sulfate-reducing conditions in marine sediments, using benzoate as a model aromatic compound. Here, in anaerobic incubations of sediments from the Pearl River Estuary, the addition of hematite or magnetite (20 mM as Fe atom) enhanced the rates of sulfate-dependent benzoate degradation by 81.8 and 91.5%, respectively, compared with control incubations without iron oxides. Further experiments demonstrated that the rate of sulfate-dependent benzoate degradation accelerated with increased magnetite concentration (5, 10, and 20 mM). The detection of acetate as an intermediate product implied syntrophic benzoate degradation pathway, which was also supported by the abundance of putative acetate- or/and H2-utilizing sulfate reducers from microbial community analysis. Microbial reduction of iron oxides under sulfate-reducing conditions only accounted for 2–11% of electrons produced by benzoate oxidation, thus the stimulatory effect of conductive iron oxides on sulfate-dependent benzoate degradation was not mainly due to an increased pool of terminal electron acceptors. The enhanced rates of syntrophic benzoate degradation by the presence of conductive iron oxides probably resulted from the establishment of a direct interspecies electron transfer (DIET) between syntrophic partners. In the presence of magnetite, Bacteroidetes and Desulfobulbaceae with potential function of extracellular electron transfer might be involved in syntrophic benzoate degradation. Results from this study will contribute to the development of new strategies for in situ bioremediation of anaerobic sediments contaminated with aromatic compounds, and provide a new perspective for the natural attenuation of aromatic compounds in iron-rich marine sediments.

Bacterial Dispersal Promotes Biodegradation in Heterogeneous Systems Exposed to Osmotic Stress.

Contaminant biodegradation in soils is hampered by the heterogeneous distribution of degrading communities colonizing isolated microenvironments as a result of the soil architecture. Over the last years, soil salinization was recognized as an additional problem especially in arid and semiarid ecosystems as it drastically reduces the activity and motility of bacteria. Here, we studied the importance of different spatial processes for benzoate biodegradation at an environmentally relevant range of osmotic potentials (ΔΨo) using model ecosystems exhibiting a heterogeneous distribution of the soil-borne bacterium Pseudomonas putida KT2440. Three systematically manipulated scenarios allowed us to cover the effects of (i) substrate diffusion, (ii) substrate diffusion and autonomous bacterial dispersal, and (iii) substrate diffusion and autonomous as well as mediated bacterial dispersal along glass fiber networks mimicking fungal hyphae. To quantify the relative importance of the different spatial processes, we compared these heterogeneous scenarios to a reference value obtained for each ΔΨo by means of a quasi-optimal scenario in which degraders were ab initio homogeneously distributed. Substrate diffusion as the sole spatial process was insufficient to counteract the disadvantage due to spatial degrader heterogeneity at ΔΨo ranging from 0 to −1 MPa. In this scenario, only 13.8−21.3% of the quasi-optimal biodegradation performance could be achieved. In the same range of ΔΨo values, substrate diffusion in combination with bacterial dispersal allowed between 68.6 and 36.2% of the performance showing a clear downwards trend with decreasing ΔΨo. At −1.5 MPa, however, this scenario performed worse than the diffusion scenario, possibly as a result of energetic disadvantages associated with flagellum synthesis and emerging requirements to exceed a critical population density to resist osmotic stress. Network-mediated bacterial dispersal kept biodegradation almost consistently high with an average of 70.7 ± 7.8%, regardless of the strength of the osmotic stress. We propose that especially fungal network-mediated bacterial dispersal is a key process to achieve high functionality of heterogeneous microbial ecosystems also at reduced osmotic potentials. Thus, mechanical stress by, for example, soil homogenization should be kept low in order to preserve fungal network integrity.

Mycelium-Like Networks Increase Bacterial Dispersal, Growth, and Biodegradation in a Model Ecosystem at Various Water Potentials

Fungal mycelia serve as effective dispersal networks for bacteria in water-unsaturated environments, thereby allowing bacteria to maintain important functions, such as biodegradation. However, poor knowledge exists on the effects of dispersal networks at various osmotic (Ψo) and matric (Ψm) potentials, which contribute to the water potential mainly in terrestrial soil environments. Here we studied the effects of artificial mycelium-like dispersal networks on bacterial dispersal dynamics and subsequent effects on growth and benzoate biodegradation at ΔΨo and ΔΨm values between 0 and -1.5 MPa. In a multiple-microcosm approach, we used a green fluorescent protein (GFP)-tagged derivative of the soil bacterium Pseudomonas putida KT2440 as a model organism and sodium benzoate as a representative of polar aromatic contaminants. We found that decreasing ΔΨo and ΔΨm values slowed bacterial dispersal in the system, leading to decelerated growth and benzoate degradation. In contrast, dispersal networks facilitated bacterial movement at ΔΨo and ΔΨm values between 0 and -0.5 MPa and thus improved the absolute biodegradation performance by up to 52 and 119% for ΔΨo and ΔΨm, respectively. This strong functional interrelationship was further emphasized by a high positive correlation between population dispersal, population growth, and degradation. We propose that dispersal networks may sustain the functionality of microbial ecosystems at low osmotic and matric potentials.

Promoter fusions with optical outputs in individual cells and in populations.

Reporter genes are widely used to quantify promoter activity, which controls production of mRNA through the interplay with RNA polymerases and transcription factors. Some of such reporters have either diffuse (lux) or focused (GFP) optical outputs that allow description of transcriptional activity in populations and in single cells. This chapter discusses the use of a dual reporter system GFP-luxCDABE that is placed in broad-host-range plasmids having origins of replication from RK2 and pBBR1. The value of this system is shown in Pseudomonas putida by characterizing the activity of the Pb promoter, which drives an operon for benzoate biodegradation in this bacterium. To this end we compare in the same cells bioluminescence as the output signal of the whole population and single cell-bound fluorescence caused by GFP expression and revealed by flow cytometry assays.

Biodegradation of benzoate by protoplast fusant via intergeneric protoplast fusion between Pseudomonas putida and Bacillus subtili

Intergeneric protoplast fusion was performed between Pseudomonas putida, a strain with moderate benzoate tolerance and degradation ability (<2000 mg l−1) while slow growth rate (specific growth rate μ = 4.5 × 10−3 h−1) and Bacillus subtilis, a strain with fast growth rate while intolerance to benzoate. A stable fusant (F29) with enhanced benzoate tolerance (as high as 10 000 mg l−1) and degradation ability (μ = 30.9 × 10−3 h−1) was obtained. The fusant is similar to P. putida in terms of amplified 16S ribosomal DNA restriction analysis (16S-ARDRA) and 16S rDNA clone sequencing analysis while alike to B. subtilis according to Gram staining. Possible biodegradation pathway of benzoate by F29 is benzene ring cleavage between two hydroxyl groups (ortho cleavage) with catechol being intermediate metabolite. However, the verification of benzoate biodegradation mechanism by this fusant requires further investigation.

Biodegradation by activated sludge and toxicity of tetracycline into a semi-industrial membrane bioreactor

Much attention has been devoted recently to the fate of pharmaceutically active compounds such as tetracycline antibiotics in soil and water. Tetracycline (TC) biodegradability by activated sludge derived from membrane bioreactor (MBR) treating swine wastewater via CO 2-evolution was evaluated by means of modified Sturm test, which was also used to evaluate its toxicity on carbon degradation. The impact of tetracycline on a semi-industrial MBR process was also examined and confronted to lab-scale experiments. After tetracycline injection in the pilot, no disturbance was detected on the elimination of organic matters and ammonium (nitrification), reaching after injection 88% and 99% respectively; only denitrification was slightly affected. Confirming the ruggedness and the superiority of membrane bioreactors over conventional bioreactors, no toxicity was observed at the considered level of TC in the pilot (20 mg TOC L −1), while at lab-scale sodium benzoate biodegradation was completely inhibited from 10 mg TOC L −1 TC. The origin of the activated sludge showed a significant impact on the performances, since the ultimate biodegradation was in the range −50% to −53% for TC concentrations in the range 10–20 mg TOC L −1 with conventional bioreactor sludge and increased to 18% for 40 mg TOC L −1 of TC with activated sludge derived from the MBR pilot. This confirmed the higher resistance of activated sludge arising from membrane bioreactor.

Catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate with a proteomics approach

The catabolic pathways and cellular responses of Pseudomonas putida P8 during growth on benzoate were studied through proteomics approach. Two-dimensional gel electrophoresis (2-DE) gel profiles of P. putida cells grown on 100 and 800 mg/L benzoate were quantitatively compared using threshold criteria and statistical tools. Protein spots of interest were identified through database searching based on peptide mass fingerprints (PMFs) obtained using matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Eight catabolic enzymes involved in both the ortho-cleavage (CatB, PcaI, and PcaF) and the meta-cleavage (DmpC, DmpD, DmpE, DmpF, and DmpG) pathways for benzoate biodegradation were identified in P. putida grown on 800 mg/L of benzoate while no meta-cleavage pathway enzymes were observed in the 2-DE gel profiles of P. putida grown on 100 mg/L of benzoate. The activation of both the ortho- and the meta-cleavage pathways in P. putida P8 grown on high benzoate concentration was confirmed directly at the protein level. In addition, another 28 differentially expressed proteins were also identified, including proteins involved in (i) detoxification and stress response (AhpC, ATPase-like ATP-binding region, putative DNA-binding stress protein, SodB and catalase/peroxidase HPI); (ii) carbohydrate, amino acid/protein and energy metabolism (isocitrate dehydrogenase, SucC, SucD, AcnB, GabD, ArcA, ArgI, Efp and periplasmic binding proteins of several ABC-transporters); and (iii) cell envelope and cell division (bacterial surface antigen family protein and MinD). Based on the data obtained, physiological changes of P. putida in response to growth on benzoate at different concentrations were discussed.

Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.

Biodegradation of sodium benzoate by a Gram-negative consortium in a laboratory-scale fluidized bed bioreactor

Gram-negative bacteria with the potential to metabolize n-alkanes and cyclic hydrocarbons were isolated from local soils and identified using 16S rDNA sequence analysis. Three isolates (CS1CO, GL1CO, GCI1CO) were identified as strains of Pseudomonas ( P.) aeruginosa and a further strain (DSS2) as P. putida. Isolates were co-cultured in a laboratory-scale fluidized bed biofilm bioreactor (FBBR) utilizing sodium benzoate as the sole carbon source, under two batch and/or one continuous growth conditions. Biofilm and planktonic bacterial growth dynamics were monitored by plate counts, and optical density measurements (230 nm) determined benzoate biodegradation. Overall higher attached and planktonic bacterial counts, and benzoate depletion, were determined under batch compared to continuous conditions, and the bioreactor performed better during the second batch phase when compared to the first batch phase. It thus appeared that both the planktonic and biofilm components of the system were necessary for the most successful sodium benzoate degradation in this system.

KINETICS OF PHENOL AND BENZOATE BIODEGRADATION IN STATIC CULTIVATION SYSTEM BY Burkholderia cepacia G4.

Kinetics of phenol and benzoate degradation by Burkholderia cepacia G4 in classical batch mode of static culture were investigated over a wide range of initial substrate concentrations applied as single substrates. Due to better adaptation to changing conditions, faster degradation of both phenol and benzoate was observed when higher concentrations were used. The results show decrease of the biomass yield coefficient, YX/S, from 0.83 to 0.31 g g-1 when the initial phenol concentration was increased from 0.54 to 0.73 g L-1, supporting the well known inhibitory effect of phenol. On the other hand, linear increasing of the yield coefficient was observed with increasing benzoate concentration. During all experiments, maximum specific substrate consumption rates, rSmax, were reached to 0.27 g g-1h-1 for phenol and to 0.40 g g-1h-1 when benzoate was the sole source of carbon in the bioreactor. In the present study, minor inhibitory effect of benzoate was observed during their investigated concentration range. To explain their degradation modelling, experimental data of both phenol and benzoate biodegradation were fitted using various kinetic models. The results demonstrated that the Yano and Koga equation gave the best fit, compared with the other models. It means that biodegradation of phenol and benzoate can be described by the same kinetic model. Based on the kinetic data, all experiments were not sensitive to change in the saturation constant, KS; therefore, KS-value was fixed at 0.042 and 0.068 g L-1 for phenol and benzoate, respectively. After that, rSmax and the inhibition constant, Ki, parameters were re-fitted in all experiments. In general, the kinetic parameters of both phenol and benzoate degradation were influenced by the initial substrate and bacterial cell mass concentrations, in addition to adaptation with changing in the culture conditions.

The Effect of Cyclic Aerobic–Anoxic Conditions on Biodegradation of Benzoate

The response of a mixed microbial culture to cyclic aerobic and anoxic (denitrifying) conditions was studied in a chemostat with a 48-hour hydraulic residence time receiving a feed containing benzoate and pyruvate. When the cyclic conditions were 3-hour aerobic and 9-hour anoxic, the bacteria-degraded benzoate aerobically via the catechol 2,3-dioxygenase (C23DO) pathway. The quantity of C23DO remained constant throughout the anoxic period but decreased during the initial portion of the aerobic period before returning to the level present in the anoxic period. Anoxic biodegradation of benzoate was via benzoyl-CoA reductase, which remained constant regardless of the redox condition. The aerobic benzoate uptake capability (AeBUC) of the culture increased during the aerobic period but decreased during the anoxic period. The anoxic benzoate uptake capability (AnBUC) exhibited the opposite response. When the cycle was 6-hour aerobic and 6-hour anoxic, aerobic biodegradation of benzoate proceeded via the protocatechuate 4,5-dioxygenase (P45DO) pathway. The P45DO activity decreased early in the aerobic period, but then increased to the level present during the anoxic period. The level of benzoyl-CoA reductase was constant throughout the cycle. Furthermore, AeBUC and AnBUC responded in much the same way as in the 3/9-hour chemostat. During a 9-hour aerobic and 3-hour anoxic cycle, the culture synthesized both P45DO and C23DO, with the former having significantly higher activity. Unlike the other two cycles, AeBUC changed little during the aerobic period, although AnBUC decreased. The culture was well-adapted to the cyclic conditions as evidenced by the lack of accumulation of either substrate during any cycle tested. This suggests that cyclic aerobic-anoxic processes can be used in industrial wastewater-treatment facilities receiving significant quantities of simple aromatic compounds like benzoate. However, the results showed that the kinetics of benzoate degradation were different under aerobic and anoxic conditions, a situation that must be considered when modeling cyclic bioreactors receiving aromatic compounds.

Effects of oxygen on biodegradation of benzoate and 3-chlorobenzoate in a denitrifying chemostat

A mixed microbial culture degraded a mixture of benzoate (863 mg/L), 3-chlorobenzoate (3-CB) (69.7 mg/L), and pyruvate (244 mg/L) under denitrifying conditions in a chemostat. Biodegradation under denitrifying conditions was stable, complete (effluent concentrations below detection limits), and proceeded without the production of toxic intermediates like chlorocatechols. The addition of oxygen at mass input rates of 6.2%, 15.5%, and 43.9% of the mass input rate of chemical oxygen demand (COD) (337 mg COD/h) did not induce the synthesis of aerobic biodegradation pathways and thus did not disrupt biodegradation. Rather, the oxygen was used as a terminal electron acceptor, displacing a stoichiometric amount of nitrate, leading to microaerobic conditions (dissolved oxygen concentration <0.050 mg/L) in which oxygen utilization and denitrification occurred simultaneously. The reduction of nitrate occurred fully to N(2) gas with no accumulation of nitrite, nitrous oxide, or nitric oxide, although the ability of the culture to transfer electrons to the nitrogen oxides decreased as the oxygen input was increased. The anoxic benzoate uptake capability was unaffected by the increase in oxygen addition, but the anoxic 3-CB uptake capability increased, as did the level of benzoyl-CoA reductase in the cells.

The impact of feed composition on biodegradation of benzoate under cyclic (aerobic/anoxic) conditions

The response of a mixed microbial culture to different feed compositions, that is, containing benzoate and pyruvate as sole carbon sources at different levels, was studied in a chemostat with a 48-h hydraulic residence time under cyclic aerobic and anoxic (denitrifying) conditions. The cyclic bacterial culture was well adapted to different feed compositions as evidenced by the lack of accumulation of benzoate or pyruvate in the chemostat. Both the benzoate-degrading capabilities and the in vitro catechol 2,3-dioxygenase (C23DO) activities of the cyclic bacterial cultures were in direct proportion to the flux through the chemostat of the substrate degraded by the pathway containing C23DO, with some exceptions. The quantity of C23DO showed a transient decrease during the initial portion of the aerobic period before returning to the level present during the anoxic period. That decrease was most likely caused by the production of H 2O 2 by the cells upon being returned to aerobic conditions.

Effects of oxygen on anoxic biodegradation of benzoate during continuous culture.

In this study, various amounts of oxygen were added to denitrifying chemostats receiving benzoate to mimic the input of oxygen to anoxic zones of biological nutrient removal systems. The effect of oxygen on the biodegradative capability of the mixed-microbial culture for benzoate was investigated. The anoxic benzoate biodegradative capability of the culture was not significantly changed as the mass flowrate of oxygen was increased to 40% of the input benzoate chemical oxygen demand (COD) mass flowrate, but was decreased approximately 70% when the mass flowrate of oxygen was increased to 70% of the input benzoate COD mass flowrate. The decrease in the anoxic benzoate biodegradative capability was due primarily to the loss of the denitrifying enzymes (measured by the anoxic pyruvate-degrading ability) and not to the loss of the key anoxic catabolic enzyme (benzoyl-coenzyme A reductase). The proportional increase in the concentration of nitrate as the residual terminal electron acceptor and the lack of synthesis of aerobic ring-cleavage enzymes as the oxygen input to the chemostat was increased suggest that the mixed microbial culture preferred oxygen to nitrate as the terminal electron acceptor, but degraded benzoate using the anoxic metabolic pathway. The concentration of the mixed microbial culture increased as the oxygen input to the chemostat was increased, suggesting that the oxygen was used by cytochrome cbb3 rather than quinol oxidase because the energetic yield of cytochrome cbb3 is higher than that of quinol oxidase or the nitrogen oxide reductases.

Use of tracer tests to evaluate the impact of enhanced-solubilization flushing on in-situ biodegradation

Tracer tests were conducted to evaluate the effect of a complexing sugar flush (CSF) on in-situ biodegradation potential at a site contaminated by jet fuel, solvents, and other organic compounds. Technical-grade hydroxypropyl-β-cyclodextrin was used during the CSF study, which was conducted in a hydraulically isolated cell emplaced in a surficial aquifer. In-situ biodegradation potential was assessed with the use of tracer tests, which were conducted prior to and immediately following the CSF study. Ethanol, hexanol, and benzoate were used as the biodegradable tracers, while bromide was used as a nonreactive tracer. The results indicate that the biodegradation of benzoate was similar for both tracer tests. Conversely, the biodegradation of ethanol (23% increase) and hexanol (41% increase) was greater for the post-CSF tracer test. In addition, analysis of core samples collected from within the test cell indicates that the population density of aerobic jet-fuel degraders increased in the vicinity of the injection wells during the CSF. These results indicate that the cyclodextrin flush did not deleteriously affect the indigenous microbial community. This study illustrates that tracer tests can be used to evaluate the impact of remediation activities on in-situ biodegradation potential.

Stochastic-convective transport with nonlinear reaction and mixing: application to intermediate-scale experiments in aerobic biodegradation in saturated porous media

Aerobic biodegradation of benzoate by Pseudomonas cepacia sp. in a saturated heterogeneous porous medium was simulated using the stochastic-convective reaction (SCR) approach. A laboratory flow cell was randomly packed with low permeability silt-size inclusions in a high permeability sand matrix. In the SCR upscaling approach, the characteristics of the flow field are determined by the breakthrough of a conservative tracer. Spatial information on the actual location of the heterogeneities is not used. The mass balance equations governing the nonlinear and multicomponent reactive transport are recast in terms of reactive transports in each of a finite number of discrete streamtubes. The streamtube ensemble members represent transport via a steady constant average velocity per streamtube and a conventional Fickian dispersion term, and their contributions to the observed breakthroughs are determined by flux-averaging the streamtube solute concentrations. The resulting simulations were compared to those from a high-resolution deterministic simulation of the reactive transport, and to alternative ensemble representations involving (i) effective Fickian travel time distribution function, (ii) purely convective streamtube transport, and (iii) streamtube ensemble subset simulations. The results of the SCR simulation compare favorably to that of a sophisticated high-resolution deterministic approach.