Biodegradation Of Benzene Research Articles

Biodegradation of Naphthalene, Benzene, Toluene, Ethyl Benzene, and Xylene in Batch and Membrane Bioreactors

Abstract Produced water is the liquid waste that is generated during coal bed methane and oil explorations. Treatment and management of produced water pose a great challenge to the sustainable operation of oil, gas, and coal bed methane operations. Proper treatment of produced water leading to its reuse can provide a sustainable water management strategy for produced water. Presence of polyaromatic hydrocarbons and benzene, toluene, ethyl benzene, and xylene (BTEX) compounds in produced water is of particular concern because these contaminants are recalcitrant and slow to degrade. In this article, we report the results of biological treatment of these compounds in laboratory-scale batch and membrane bioreactors. Naphthalene was considered a model compound to represent polyaromatic hydrocarbons. The strategy employed involves the enrichment of bacteria capable of degrading naphthalene and BTEX in separate batch reactors and study of their degradation kinetics. A membrane bioreactor was initiated with seed ...

A Bench-Scale Constructed Wetland As a Model to Characterize Benzene Biodegradation Processes in Freshwater Wetlands

In wetlands, a variety of biotic and abiotic processes can contribute to the removal of organic substances. Here, we used compound-specific isotope analysis (CSIA), hydrogeochemical parameters and detection of functional genes to characterize in situ biodegradation of benzene in a model constructed wetland over a period of 370 days. Despite low dissolved oxygen concentrations (<30 μM), the oxidation of ammonium to nitrate and the complete oxidation of ferrous iron pointed to a dominance of aerobic processes, suggesting efficient oxygen transfer into the sediment zone by plants. As benzene removal became highly efficient after day 231 (>98% removal), we applied CSIA to study in situ benzene degradation by indigenous microbes. Combining carbon and hydrogen isotope signatures by two-dimensional stable isotope analysis revealed that benzene was degraded aerobically, mainly via the monohydroxylation pathway. This was additionally supported by the detection of the BTEX monooxygenase gene tmoA in sediment and root samples. Calculating the extent of biodegradation from the isotope signatures demonstrated that at least 85% of benzene was degraded by this pathway and thus, only a small fraction was removed abiotically. This study shows that model wetlands can contribute to an understanding of biodegradation processes in floodplains or natural wetland systems.

Substrate interactions during the biodegradation of BTEX and THF mixtures by Pseudomonas oleovorans DT4

The efficient tetrahydrofuran (THF)-degrading bacterium, Pseudomonas oleovorans DT4 was used to investigate the substrate interactions during the aerobic biotransformation of THF and BTEX mixtures. Benzene and toluene could be utilized as growth substrates by DT4, whereas cometabolism of m-xylene, p-xylene and ethylbenzene occurred with THF. In binary mixtures, THF degradation was delayed by xylene, ethylbenzene, toluene and benzene in descending order of inhibitory effects. Conversely, benzene (or toluene) degradation was greatly enhanced by THF leading to a higher degradation rate of 39.68 mg/(h g dry weight) and a shorter complete degradation time about 21 h, possibly because THF acted as an “energy generator”. Additionally, the induction experiments suggested that BTEX and THF degradation was initiated by independent and inducible enzymes. The transient intermediate hydroquinone was detected in benzene biodegradation with THF while catechol in the process without THF, suggesting that P. oleovorans DT4 possessed two distinguished benzene pathways.

Microbial Biodegradation of Aromatic Compounds in a Soil Contaminated with Gasohol

The studies developed in this work aimed to find alternatives to biodegradation or bioremediation of soils contaminated with gasoline or gasohol. So, the biodegradation of benzene, toluene and o-xylene (BTX) in soil samples contaminated with gasoline or gasohol by a bacterial consortium was studied. Four bacterial strains were selected for the consortium based on their growth capacity in gasoline, gasohol and BTX as sole carbon sources, and on the production of biosurfactants in mineral medium containing gasohol as the sole carbon source. The reduction of TX concentrations in soil slurries in a multi-cell bioreactor system was used as the criterion to evaluate biodegradation efficiency. BTX removal was highly stimulated by air injection and mineral nutrients, and was significantly increased by the presence of the bacterial consortium. Addition of a proprietary oxygen release compound did not stimulate the biodegradation of BTX.

Substrate interactions during the biodegradation of benzene and toluene hydrocarbons at various salinities

Substrate interactions during the biodegradation of benzene and toluene hydrocarbons at various salinities

Degradation of BTEX by anaerobic bacteria: physiology and application

Pollution of the environment with aromatic hydrocarbons, such as benzene, toluene, ethylbenzene and xylene (so-called BTEX) is often observed. The cleanup of these toxic compounds has gained much attention in the last decades. In situ bioremediation of aromatic hydrocarbons contaminated soils and groundwater by naturally occurring microorganisms or microorganisms that are introduced is possible. Anaerobic bioremediation is an attractive technology as these compounds are often present in the anoxic zones of the environment. The bottleneck in the application of anaerobic techniques is the lack of knowledge about the anaerobic biodegradation of benzene and the bacteria involved in anaerobic benzene degradation. Here, we review the existing knowledge on the degradation of benzene and other aromatic hydrocarbons by anaerobic bacteria, in particular the physiology and application, including results on the (per)chlorate stimulated degradation of these compounds, which is an interesting new alternative option for bioremediation.

Removal of benzene under acidic conditions in a controlled Trickle Bed Air Biofilter

Trickle Bed Air Biofilters (TBABs) are considered to be economical and environmental-friendly for treatment of Volatile Organic Compounds (VOCs). Hydrophilic VOCs are easily degradable while hydrophobic ones pose a great challenge for adequate treatment due to the transfer of the VOC to the liquid phase. In this study the utilization of acidic pH is proposed for the treatment of benzene vapors. The acidic pH would encourage the growth of fungi as the main consortium. A TBAB operated at pH 4 was used for the treatment of an air stream contaminated with benzene under different loading rates ranging from 37 to 76.8g/(m3h). The purpose of introducing fungi was to compare the performance with traditional TBAB operating under neutral pH in order to assess the biodegradation of benzene in mixtures with other compounds favoring acidic conditions. The experimental plan was designed to assess long-term performance with emphasis based on different benzene loading rates, removal efficiency with TBAB depth, and carbon mass balance closure. At benzene loading rate of 64g/(m3h), the removal efficiency was 90%. At the maximum loading rate of 77g/(m3h), the removal efficiency was 75% marking the maximum elimination capacity for the TBAB at 58.8g/(m3h). Operating at acidic pH successfully supported the degradation of benzene in TBAB. It is worthwhile to note that benzene appears in mixtures with n-hexane and toluene, which are reported to be better degraded under such conditions.

Design methodology accounting for the effects of porous medium heterogeneity on hydraulic residence time and biodegradation in horizontal subsurface flow constructed wetlands

Horizontal flow constructed wetlands are engineered systems capable of eliminating a wide range of pollutants from the aquatic environment. Nevertheless, poor hydrodynamic behavior is commonly found resulting in preferential pathways and variations in both (i) the hydraulic residence time distribution (HRTD) and, consequently, (ii) the wetland's treatment efficiency. The aim of this work was to outline a methodology for wetland design that accounts for the effect of heterogeneous hydraulic properties of the porous substrate on the HRTD and treatment efficiency. Biodegradation of benzene was used to illustrate the influence of hydraulic conductivity heterogeneity on wetland efficiency. Random, spatially correlated hydraulic conductivity fields following a log-normal distribution were generated and then introduced in a subsurface flow numerical model. The results showed that the variance of the distribution and the correlation length in the longitudinal direction are key indicators of the extent of heterogeneity. A reduction of the mean hydraulic residence time was observed as the extent of heterogeneity increased, while the HRTD became broader with increased skewness. At the same time, substrate heterogeneity induced preferential flow paths within the wetland bed resulting in variations of the benzene treatment efficiency. Further to this it was observed that the distribution of biomass within the porous bed became heterogeneous, rising questions on the representativeness of sampling. It was concluded that traditional methods for wetland design based on assumptions such as a homogeneous porous medium and plug flow are not reliable. The alternative design methodology presented here is based on the incorporation of heterogeneity directly during the design phase. The same methodology can also be used to optimize existing systems, where the HRTD has been characterized with tracer experiments.

Novel aerobic benzene degrading microorganisms identified in three soils by stable isotope probing

The remediation of benzene contaminated groundwater often involves biodegradation and although the mechanisms of aerobic benzene biodegradation in laboratory cultures have been well studied, less is known about the microorganisms responsible for benzene degradation in mixed culture samples or at contaminated sites. To address this knowledge gap, DNA based stable isotope probing (SIP) was utilized to identify active benzene degraders in microcosms constructed with soil from three sources (a contaminated site and two agricultural sites). For this, replicate microcosms were amended with either labeled (¹³C) or unlabeled benzene and the extracted DNA samples were ultracentrifuged, fractioned and subject to terminal restriction fragment length polymorphism (TRFLP). The dominant benzene degraders (responsible for ¹³C uptake) were determined by comparing relative abundance of TRFLP phylotypes in heavy fractions of labeled benzene (¹³C) amended samples to the controls (from unlabeled benzene amended samples). Two phylotypes (a Polaromonas sp. and an Acidobacterium) were the major benzene degraders in the microcosms constructed from the contaminated site soil, whereas one phylotype incorporated the majority of the benzene-derived ¹³C in each of the agricultural soils ("candidate" phylum TM7 and an unclassified Sphingomonadaceae).

Biodegradation of benzene by pure and mixed cultures of Bacillus spp.

Benzene has a wide range of industrial applications, but it is also a major source of environmental pollution. The most eco-friendly/cost-effective method of remediation is biodegradation. In the present study, we used a variety of microbial strains in different combinations on a selection of substrate concentrations to determine the most effective degradation processes. Bacterial strains of pure culture (L4, N3, and N6) were isolated from oil sludge in both Luria–Bertani buffer (LB) and nutrient broth media, and identified by 16S-rRNA analysis (≥98% similarity). The degradation experiments were performed using different combinations of bacterial strains (L4, N3, N6, L4 + N3, L4 + N6, N3 + N6, and L4 + N3 + N6) in modified carbon-free media with different concentrations of benzene as a carbon source (60, 100, and 160 mg l−1) at 30 °C. The isolates of L4 (Acc no: FJ686821), N3 (FJ686825) and N6 (FJ868628) were identified as Bacillus spp. using 16S-rRNA gene sequence analysis. All combinations of isolates have the capacity to degrade benzene. However, the L4 + N3 combination was more efficient than the other mixed or single cultures. In the presence of N6 isolate, the degradation rate of benzene decreased, possibly due to inter- and/or intra species interaction amongst the bacteria. The kinetic parameters ‘Km’ of the Lineweaver–Burk regressions conducted as part of this experiment showed that the lower the level of Km was, the better the biodegradation achieved. The results of this study showed that the use of Bacillus strains in benzene decomposition is feasible. In addition, different strain combinations exhibited different degradation patterns, which are attributed to the most efficient mixed cultures of Bacillus spp.

Modeling in situ benzene bioremediation in the contaminated Liwa aquifer (UAE) using the slow-release oxygen source technique

Dissolved benzene was detected in the shallow unconfined Liwa aquifer, UAE, which represents the main freshwater source for the nearby residence Bu Hasa camp area. The main source of this contamination is believed to be the rejected water released from Bu Hasa liquid recovery plant. In this paper, a finite element model (METABIOTRANS) is used to simulate the fate and transport of the dissolved benzene plume in Liwa aquifer. Different remediation scenarios were simulated in which the slow-release oxygen source (SOS) technique is utilized to minimize benzene concentrations at the nearest camp supply wells downstream of the contamination zone. Results of the remediation scenarios show that the highest biodegradation rates occur when the oxygen source is placed near the plume center; where higher benzene concentrations exist. The nearest oxygen release source to the contamination zone caused higher stimulation to bacterial growth than further down-gradient oxygen sources. It also exhibited longer resident time of oxygen in the aquifer; and therefore, yielded higher reductions in benzene concentrations. However, using one central SOS proved to be insufficient as contaminant escaped laterally. Additional four transverse oxygen sources were necessary to capture benzene that laterally spread away from the contamination zone. These lateral SOSs were essential to reduce benzene concentrations at the supply wells that are located at the plume fringes. Finally, it was found that increasing oxygen release from one source did not always improve remediation; and that using several SOSs with lower release rates could be a more practical approach to enhance benzene biodegradation in the aquifer.

Soil Physical Constraints on Intrinsic Biodegradation of Petroleum Vapors in a Layered Subsurface

Naturally occurring biodegradation of petroleum hydrocarbons in the vadose zone depends on the physical soil environment influencing field-scale gas exchange and pore-scale microbial metabolism. In this study, we evaluated the effect of soil physical heterogeneity on biodegradation of petroleum vapors in a 16-m-deep, layered vadose zone. Soil slurry experiments (soil/water ratio 10:30 w/w, 25°C) on benzene biodegradation under aerobic and well-mixed conditions indicated that the biodegradation potential in different textured soil samples was related to soil type rather than depth, in the order: sandy loam > fine sand > limestone. Similarly, O(2) consumption rates during in situ respiration tests performed at the site were higher in the sandy loam than in the fine sand, although the difference was less significant than in the slurries. Laboratory and field data generally agreed well and suggested a significant potential for aerobic biodegradation, even with nutrient-poor and deep subsurface conditions. In slurries of the sandy loam, the biodegradation potential declined with increasing in situ water saturation (i.e., decreasing air-filled porosity in the field). This showed a relation between antecedent undisturbed field conditions and the slurry biodegradation potential, and suggested airfilled porosity to be a key factor for the intrinsic biodegradation potential in the field.

Anaerobic benzene biodegradation by a pure bacterial culture of Bacillus cereus under nitrate reducing conditions

A pure culture using benzene as sole carbon and energy sources was isolated by screening procedure from gasoline contaminated soil. The analysis of the 16S rDNA gene sequence, morphological and physiological characteristics showed that the isolated strain was a member of genus Bacillus cereus. The biodegradation performance of benzene by B. cereus was evaluated, and the results showed that benzene could be efficiently biodegraded when the initial benzene concentration was below 150 mg/L. The metabolites of anaerobic nitrate-dependent benzene oxidation by strain B. cereus were identified as phenol and benzoate. The results of substrate interaction between binary combinations for benzene, phenol and benzoate showed that the simultaneous presence of benzene stimulated the degradation of benzoate, whereas the addition of benzene inhibited the degradation of phenol. Benzene degradation by B. cereus was enhanced by the addition of phenol and benzoate, the enhanced effects were more pronounced at higher concentration. To our knowledge, this is the first report that the isolated bacterial culture of B. cereus can efficiently degraded benzene under nitrate reducing conditions.

Use of bio‐traps with multiple substrates and electron acceptors to optimize remediation

AbstractBio‐Traps® were used to investigate biodegradation of benzene, methyl tertiary butyl ether (MTBE), and tertiary butyl alcohol (TBA) under different conditions at a fractured rock site to aid the selection of a bioremediation approach. The Bio‐Traps were amended with the 13C‐labeled constituent of interest and sampled sequentially at 15‐, 30‐, 60‐, and 90‐day intervals. The conditions tested were biodegradation during operation of an air sparge system, amendment with nitrate during the air sparge operation, anaerobic biodegradation with the system turned off, and anaerobic biodegradation with nitrate amendment. There was increased biomass with nitrate amendment whether the air sparge system was on or off for all the constituents of interest. The diversity of the microbial community, determined by phospholipid fatty acid analysis, decreased with nitrate amendment as more specialized degraders were selected. The most negative indicators of potential biodegradation performance were observed with the anaerobic control. There was less biomass overall, less incorporation of 13C into biomass, and decreased membrane permeability. As testing with additional amendments continues at the site, it is not yet certain which treatment might be selected for bioremediation, but the Bio‐Trap tests thus far have identified that the in situ, natural attenuation condition is least favorable for biodegradation. © 2009 Wiley Periodicals, Inc.

Benzene bioremediation using cow dung microflora in two phase partitioning bioreactor

Bioremediation of benzene has been carried out using cow dung microflora in a bioreactor. The bioremediation of benzene under the influence of cow dung microflora was found to be 100% and 67.5%, at initial concentrations of 100 mg/l and 250 mg/l within 72 h and 168 h respectively; where as at higher concentration (500 mg/l), benzene was found to be inhibitory. Hence the two phase partitioning bioreactor (TPPB) has been designed and developed to carryout biodegradation at higher concentration. In TPPB 5000 mg/l benzene was biodegraded up to 50.17% over a period of 168 h. Further the Pseudomonas putida MHF 7109 was isolated from cow dung microflora as potential benzene degrader and its ability to degrade benzene at various concentrations was evaluated. The data indicates 100%, 81% and 65% degradation at the concentrations of 50 mg/l, 100 mg/l, 250 mg/l within the time period of 24 h, 96 h and 168 h respectively. The GC-MS data also shows the presence of catechol and 2-hydroxymuconic semialdehyde, which confirms the established pathway of benzene biodegradation. The present research proves the potential of cow dung microflora as a source of biomass for benzene biodegradation in TPPB.

Carbon and hydrogen isotope fractionation of benzene during biodegradation under sulfate‐reducing conditions: a laboratory to field site approach

The microbial carbon and hydrogen isotope fractionation of benzene under sulfate-reducing conditions was investigated within systems of increasing complexity: (i) batch laboratory microcosms, (ii) a groundwater-percolated column system, and (iii) an aquifer transect. Recent molecular biological studies indicate that, at least in the laboratory microcosms and the column system, benzene is degraded by similar bacterial communities. Carbon and hydrogen enrichment factors (epsilon(C), epsilon(H)) obtained from laboratory microcosms and from the column study varied significantly although experiments were performed under similar redox and temperature conditions. Thus, enrichment factors for only a single element could not be used to distinguish benzene degradation under sulfate-reducing conditions from other redox conditions. In contrast, using correlation of changes of hydrogen vs. carbon isotope ratios (Lambda = Delta delta(2)H/Delta delta(13)C), similar Lambda-values were derived for the benzene biodegradation under sulfate-reducing conditions in all three experimental systems (Lambda(laboratory microcosms) = 23 +/- 5, Lambda(column) = 28 +/- 3, Lambda(aquifer) = 24 +/- 2), showing the robustness of the two-dimensional compound-specific stable isotope analysis (2D-CSIA) for elucidating distinct biodegradation pathways. Comparing carbon and hydrogen isotope fractionation data from recent studies, an overlap in Lambda-values was observed for benzene biodegradation under sulfate-reducing (Lambda = 23 +/- 5 to Lambda = 29 +/- 3) and methanogenic (Lambda = 28 +/- 1 to Lambda = 39 +/- 5) conditions, indicating a similar initial benzene reaction mechanism for both electron-acceptor conditions.

Biodegradation of BTEX at high salinity by an enrichment culture from hypersaline sediments of Rozel Point at Great Salt Lake

The primary goal of this research was to assess the biodegradation of benzene, toluene, ethylbenzene and xylenes in sediment from Great Salt Lake, near Rozel Point, UT. An enrichment culture that degraded benzene or toluene as the sole carbon source at high salinity was developed from a sediment sample obtained from Rozel Point. The enrichment degraded benzene or toluene within 1, 2 and 5 weeks in the presence of 14%, 23% and 29% NaCl respectively. PCR studies using degenerate primers revealed that degradation occurred primarily via catechol and the meta-cleavage pathway. Molecular analysis showed that the Gammaproteobacteria were the dominant members of the enrichment and that shifts in community composition occurred during benzene metabolism. This study demonstrated that micro-organisms at Rozel Point have the ability to degrade hydrocarbons over a broad range of salinities (1-5 mol l(-1) NaCl) and that the members of the Gammaproteobacteria class play an important role in the degradation process. These results are significant as little is known about the fate of petroleum seeps at Rozel Point. Also, the identity of microbes and the key enzymes involved in the degradation steps are important for understanding natural attenuation potential of hydrocarbons.

Field metabolomics and laboratory assessments of anaerobic intrinsic bioremediation of hydrocarbons at a petroleum‐contaminated site

SummaryField metabolomics and laboratory assays were used to assess the in situ anaerobic attenuation of hydrocarbons in a contaminated aquifer underlying a former refinery. Benzene, ethylbenzene, 2‐methylnaphthalene, 1,2,4‐ and 1,3,5‐trimethylbenzene were targeted as contaminants of greatest regulatory concern (COC) whose intrinsic remediation has been previously reported. Metabolite profiles associated with anaerobic hydrocarbon decay revealed the microbial utilization of alkylbenzenes, including the trimethylbenzene COC, PAHs and several n‐alkanes in the contaminated portions of the aquifer. Anaerobic biodegradation experiments designed to mimic in situ conditions showed no loss of exogenously amended COC; however, a substantive rate of endogenous electron acceptor reduction was measured (55 ± 8 µM SO4 day−1). An assessment of hydrocarbon loss in laboratory experiments relative to a conserved internal marker revealed that non‐COC hydrocarbons were being metabolized. Purge and trap analysis of laboratory assays showed a substantial loss of toluene, m‐ and o‐xylene, as well as several alkanes (C6–C12). Multiple lines of evidence suggest that benzene is persistent under the prevailing site anaerobic conditions. We could find no in situ benzene intermediates (phenol or benzoate), the parent molecule proved recalcitrant in laboratory assays and low copy numbers of Desulfobacterium were found, a genus previously implicated in anaerobic benzene biodegradation. This study also showed that there was a reasonable correlation between field and laboratory findings, although with notable exception. Thus, while the intrinsic anaerobic bioremediation was clearly evident at the site, non‐COC hydrocarbons were preferentially metabolized, even though there was ample literature precedence for the biodegradation of the target molecules.

Two‐dimensional modelling of benzene transport and biodegradation in a laboratory‐scale aquifer

In this study biodegradation of aqueous benzene during transport in a laboratory‐scale aquifer model was investigated by conducting a 2‐D plume test and numerical modelling. Benzene biodegradation and transport was simulated with the 2‐D numerical model developed for solute transport coupled with a Haldane‐Andrews type function for inclusion of an inhibition constant which is effective for high concentrations. Experimental data revealed that in the early stages the benzene plume showed a rather clear shape but lost its shape with increased travel time. The mass recoveries of benzene at 9, 16, and 22 h were 37, 13 and 8%, respectively, showing that a significant mass reduction of aqueous benzene occurred in the model aquifer. The major processes responsible for the mass reduction were biodegradation and irreversible sorption. The modelling results also indicated that the simulation based on the microbial parameters from the batch experiments slightly overestimated the mass reduction of benzene during transport. The sensitivity analysis demonstrated that the benzene plume was sensitive to the maximum specific growth rate and slightly sensitive to the half‐saturation constant of benzene but almost insensitive to the Haldane inhibition constant. The insensitivity to the Haldane inhibition constant was due to the rapid decline of the benzene peak concentration by natural attenuation such as hydrodynamic dispersion and irreversible sorption. An analysis of the model simulation also indicated that the maximum specific growth rate was the key parameter controlling the plume behaviour, but its impact on the plume was affected by competing parameter such as the irreversible sorption rate coefficient.

Mixed aerobic and anaerobic microbial communities in benzene-contaminated groundwater

To investigate the factors affecting benzene biodegradation and microbial community composition in a contaminated aquifer. We identified the microbial community in groundwater samples from a benzene-contaminated aquifer situated below a petrochemical plant. Eleven out of twelve groundwater samples with in situ dissolved oxygen concentrations between 0 and 2.57 mg l(-1) showed benzene degradation in aerobic microcosm experiments, whereas no degradation in anaerobic microcosms was observed. The lack of aerobic degradation in the remaining microcosm could be attributed to a pH of 12.1. Three groundwaters, examined by 16S rRNA gene clone libraries, with low in situ oxygen concentrations and high benzene levels, each had a different dominant aerobic (or denitrifying) population, either Pseudomonas, Polaromonas or Acidovorax species. These groundwaters also had syntrophic organisms, and aceticlastic methanogens were detected in two samples. The alkaline groundwater was dominated by organisms closely related to Hydrogenophaga. Results show that pH 12.1 is inimical to benzene biodegradation, and that oxygen concentrations below 0.03 mg l(-1) can support aerobic benzene-degrading communities. These findings will help to guide the treatment of contaminated groundwaters, and raise questions about the extent to which aerobes and anaerobes may interact to effect benzene degradation.