Anaerobic Biodegradation of Alkylbenzenes in Laboratory Microcosms Representing Ambient Conditions

A microcosm study was performed to document the anaerobic biodegradation of benzene, toluene, ethylbenzene, m- xylene, and/or o-xylene in petroleum-contaminated aquifer sediment from sites in Michigan (MI) and North Carolina (NC) and relate the results to previous field investigations of intrinsic bioremediation. Laboratory microcosms, designed to simulate ambient conditions, were constructed under anaerobic conditions with sediment and groundwater from source, mid-plume, and end-plume locations at each site. The general patterns of biodegradation and electron acceptor utilization in the microcosms were consistent with field data. At the MI site, methane was produced after a moderate lag period, followed by toluene degradation in all sets of microcosms. At the NC site, biodegradation of the target compounds was not evident in the source area microcosms. In the mid-plume microcosms, toluene and o-xylene biodegraded first, followed by m-xylene and benzene, a pattern consistent with contaminant decay along the plume length. Chemical extraction of microcosm sediment at the beginning and end of me incubation indicated that iron-reducing conditions were dominant and iron reduction occurred on a sediment fraction not extracted by 0.5N HC1. In the end-plume microcosms, degradation of benzene, toluene, and xylene isomers occurred but was variable between replicates. Consistent with field data, dissolved concentrations of the target contaminant(s) persisted at low but detectable levels (0.05 to 0.25 μM) in microcosms from both sites where biodegradation was measured.     

Microbial Metabolism of Benzene and the Oxidation of Ferrous Iron under Anaerobic Conditions: Implications for Bioremediation

Benzene and toluene were biodegraded when chelated Fe(III) served as the terminal electron acceptor in aquifer sediments contaminated by a petroleum refinery. Benzene biodegradation ceased when Fe(III) was depleted but resumed upon reamendment. Microorganisms from the same sediments degraded toluene, but not benzene, under nitrate reducing conditions. However, the anaerobic oxidation of Fe(II) to Fe(III) was also observed in toluene-degrading incubations. Fe(II) oxidation was dependent on the presence of nitrate and enhanced when organic electron donors were provided. Microbial nitrate-linked Fe(II) oxidation was also documented in other petroleum-contaminated aquifer sediments, sludge from an oil–water separator, a landfill leachate-impacted aquifer and a garden soil. These observations suggest that some of the reported effects of nitrate on hydrocarbon biodegradation may be indirect through the reoxidation of Fe(II).     

Stimulation of anaerobic biodegradation of DDT and its metabolites in a muck soil: laboratory microcosm and mesocosm studies

The aim of this study was to evaluate the impact of selected electron donors and electron acceptors on the anaerobic biodegradation of DDT and its major metabolites in a muck soil with a long history of exposure to the pesticide. Loss of DDT was measured in anaerobic microcosms supplemented with H₂, lactate, and acetate. The greatest loss of DDT (approximately 87 %) was observed in microcosms amended with lactate and no additional electron acceptor compared to the no additional electron donor or acceptor sets. An increase in measureable concentrations of DDx was observed in un-amended microcosms. In larger scale mesocosms, significant increases in dissolved organic carbon (DOC) corresponded with low redox potentials. Increases in DOC corresponded with sharp increases in measured concentrations of DDx, followed by a decrease in measured DDT concentrations in lactate-amended mesocosms. Our studies indicate that sorbed DDx is released upon anaerobic incubation, and that indigenous microorganisms capable of DDx degradation respond to lactate additions. Both the potential for release of sorbed DDx and the potential for biodegradation of DDx should be considered during remediation of DDx-contaminated organic soils at low redox potentials.     

Geochemical and physiological evidence for mixed aerobic and anaerobic field biodegradation of coal tar waste by subsurface microbial communities

We used geochemical analyses of groundwater and laboratory-incubated microcosms to investigate the physiological responses of naturally occurring microorganisms to coal-tar-waste constituents in a contaminated aquifer. Waters were sampled from wells along a natural hydrologic gradient extending from uncontaminated (1 well) into contaminated (3 wells) zones. Groundwater analyses determined the concentrations of carbon and energy sources (pollutants or total organic carbon), final electron acceptors (oxygen, nitrate, sulfate), and metabolic byproducts (dissolved inorganic carbon [DIC], alkalinity, methane, ferrous iron, sulfide, Mn2+). In the contaminated zone of the study site, concentrations of methane, hydrogen, alkalinity, and DIC were enhanced, while dissolved oxygen and nitrate were depleted. Field-initiated biodegradation assays using headspace-free serum bottle microcosms filled with groundwater examined metabolism of the ambient organic contaminants (naphthalene, 2-methylnaphthalene, benzothiophene, and indene) by the native microbial communities. Unamended microcosms from the contaminated zone demonstrated the simultaneous degradation of several coal-tar-waste constituents at the in situ temperature (10°C). Lag phases prior to the onset of biodegradation indicated the prevalence of both aerobic and anaerobic conditions in situ. Electron acceptor-amended microcosms from the most contaminated well waters demonstrated only aerobic naphthalene degradation. Collectively, the geochemical and microbial evidence show that biodegradation of coal-tar-waste constituents occurs via both aerobic and anaerobic terminal electron accepting processes at this site.     

Potential for anaerobic bioremediation of BTEX in petroleum-contaminated aquifers

Laboratory incubations of aquifer material or enrichments derived from aquifer material as well as geochemical data have suggested that, under the appropriate conditions, BTEX components of petroleum (benzene, toluene, ethylbenzene and xylene) can be degraded in the absence of molecular oxygen with either Fe(III), sulfate, or nitrate serving as the electron acceptor. BTEX degradation under methanogenic conditions has also been observed. However, especially for benzene, the BTEX contaminant of greatest concern, anaerobic degradation is often difficult to establish and maintain in laboratory incubations. Although studies to date have suggested that naturally occurring anaerobic BTEX degradation has the potential to remove significant quantities of BTEX from petroleum-contaminated aquifers, and mechanisms for stimulating anaerobic BTEX degradation in laboratory incubations have been developed, further study of the organisms involved in this metabolism and the factors controlling their distribution and activity are required before it will be possible to design rational strategies for accelerating anaerobic BTEX degradation in contaminated aquifers. Received 21 November 1995/ Accepted in revised form 20 February 1996     

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

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

Anaerobic benzene degradation

Although many studies have indicated that benzene persists under anaerobic conditions in petroleum-contaminated environments, it has recently been documented that benzene can be anaerobically oxidized with most commonlyconsidered electron acceptors for anaerobic respiration. These include: Fe(III),sulfate, nitrate, and possibly humic substances. Benzene can also be convertedto methane and carbon dioxide under methanogenic conditions. There is evidencethat benzene can be degraded under in situ conditions in petroleum-contaminatedaquifers in which either Fe(III) reduction or methane production is the predominant terminal electron-accepting process. Furthermore, evidence from laboratory studies suggests that benzene may be anaerobically degraded in petroleum-contaminated marine sediments under sulfate-reducing conditions. Laboratory studies have suggested that within the Fe(III) reduction zone of petroleum-contaminated aquifers, benzene degradation can be stimulated with the addition of synthetic chelators which make Fe(III) more available for microbial reduction. The addition of humic substances and other compounds that contain quinone moieties can also stimulate anaerobic benzene degradation in laboratory incubations of Fe(III)-reducing aquifer sediments by providing an electron shuttle between Fe(III)-reducing microorganisms and insoluble Fe(III) oxides. Anaerobic benzene degradation in aquifer sediments can be stimulated with the addition of sulfate, but in some instances an inoculum of benzene-oxidizing,sulfate-reducing microorganisms must also be added. In a field trial, sulfate addition to the methanogenic zone of a petroleum-contaminated aquifer stimulated the growth and activity of sulfate-reducing microorganisms and enhanced benzene removal. Molecular phylogenetic studies have provided indications of what microorganisms might be involved in anaerobic benzene degradation in aquifers. The major factor limiting further understanding of anaerobic benzene degradation is the lack of a pure culture of an organism capable of anaerobic benzene degradation.     

Benzene Degradation at a Site Amended with Nitrate or Chlorate

Aromatic hydrocarbons are widespread in nature and often contribute to the pollution of soils, sediments, and groundwater. The contamination of soil with mobile aromatic compounds, generally termed BTEX (benzene, toluene, ethylbenzene, xylene) is observed at many industrial sites, especially those associated with the petrochemical industry. In situ bioremediation of sites that are contaminated with BTEX can be applied both aerobically and anaerobically. The use of anaerobic in situ bioremediation is advantageous because supply of oxygen is not needed. Nevertheless, anaerobic in situ bioremediation is less commonly used for BTEX contaminated sites. This paper describes push-pull experiments in order to stimulate the degradation of benzene by the addition of nitrate or chlorate. Deuterated benzene was subjected with nitrate-amended groundwater to the aquifer, and the mineralization was traced by the enrichment of deuterium in the groundwater. Nitrate can be used as electron acceptor, and the addition of nitrate at a site in The Netherlands resulted in partial degradation of benzene. This was demonstrated by comparing various push-pull experiments, benzene concentration measurements, stable isotope analyses of benzene and water, and modeling. Chlorate can be used for the in situ production of oxygen, followed by degradation of benzene with oxygen as electron acceptor. The addition of chlorate at the site resulted in the complete removal of benzene demonstrating a complete degradation within 4 weeks. A pull phase was not needed during this run.     

Dehalogenation and biodegradation of brominated phenols and benzoic acids under iron-reducing, sulfidogenic, and methanogenic conditions.

The anaerobic biodegradation of monobrominated phenols and benzoic acids by microorganisms enriched from marine and estuarine sediments was determined in the presence of different electron acceptors [i.e., Fe(III), SO4(2-), or HCO3-]. Under all conditions tested, the bromophenol isomers were utilized without a lengthy lag period whereas the bromobenzoate isomers were utilized only after a lag period of 23 to 64 days. 2-Bromophenol was debrominated to phenol, with the subsequent utilization of phenol under all three reducing conditions. Debromination of 3-bromophenol and 4-bromophenol was also observed under sulfidogenic and methanogenic conditions but not under iron-reducing conditions. In the bromobenzoate-degrading cultures, no intermediates were observed under any of the conditions tested. Debromination rates were higher under methanogenic conditions than under sulfate-reducing or iron-reducing conditions. The stoichiometric reduction of sulfate or Fe(III) and the utilization of bromophenols and phenol indicated that biodegradation was coupled to sulfate or iron reduction, respectively. The production of phenol as a transient intermediate demonstrates that reductive dehalogenation is the initial step in the biodegradation of bromophenols under iron- and sulfate-reducing conditions.     

Benzene Degradation Coupled with Chlorate Reduction in a Soil Column Study

Perchlorate and chlorate are electron acceptors that during reduction result in the formation of molecular oxygen. The produced oxygen can be used for activation of anaerobic persistent pollutants, like benzene. In this study chlorate was tested as potential electron acceptor to stimulate benzene degradation in anoxic polluted soil column. A chlorate amended benzene polluted soil column was operated over a period of 500 days. Benzene was immediately degraded in the column after start up, and benzene removal recovered completely after omission of chlorate or a too high influent chlorate concentration (22 mM). Mass balance calculations showed that per mole of benzene five mole of chlorate were reduced. At the end of the experiment higher loading rates were applied to measure the maximal benzene degradation rate in this system; a breakthrough of benzene was not observed. The average benzene degradation rate over this period was 31 μmol l⁻¹ h⁻¹ with a maximal of 78 μmol l⁻¹ h⁻¹. The high degradation rate and the necessity of chlorate indicate that oxygen produced during chlorate reduction indeed is used for the activation of benzene. This is the first column study where benzene biodegradation at a high rate coupled with anaerobic chlorate reduction is observed.     

Anaerobic degradation of No. 2 diesel fuel in the wetland sediments of Barataria-Terrebonne estuary under various electron acceptor conditions

The biodegradation of No. 2 diesel fuel under anaerobic conditions was investigated using sediments collected from wetlands of Barataria-Terrebonne estuary in Louisiana. The results indicated enhanced biodegradation of diesel fuel under sulfate-reducing, nitrate-reducing, methanogenic, and mixed electron acceptor conditions. However, the rate of diesel degradation was the highest under mixed electron acceptor conditions followed in order by sulfate-reducing, methanogenic, and nitrate-reducing conditions. Under mixed electron acceptor condition, 99% removal of diesel fuel was achieved within 510 days, while under sulfate-reducing condition 62% degradation of diesel fuel was observed for the same period. Diesel fuel was also degraded to a smaller extent in the culture condition where electron acceptors were not supplemented (natural attenuation condition). This study showed evidence for enhanced diesel fuel metabolism in a mixed microbial population system similar to any contaminated field site, where a heterogeneous microbial population exists.     

RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) Biodegradation in Aquifer Sediments under Manganese-Reducing Conditions

A shallow, RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)-contaminated aquifer at Naval Submarine Base Bangor has been characterized as predominantly manganese-reducing, anoxic with local pockets of oxic conditions. The potential contribution of microbial RDX degradation to localized decreases observed in aquifer RDX concentrations was assessed in sediment microcosms amended with [U-¹⁴C] RDX. Greater than 85% mineralization of ¹⁴C-RDX to ¹⁴CO₂ was observed in aquifer sediment microcosms under native, manganese-reducing, anoxic conditions. Significant increases in the mineralization of ¹⁴C-RDX to ¹⁴CO₂ were observed in anoxic microcosms under NO₃-amended or Mn(IV)-amended conditions. No evidence of ¹⁴C-RDX biodegradation was observed under oxic conditions. These results indicate that microbial degradation of RDX may contribute to natural attenuation of RDX in manganese-reducing aquifer systems.     

Effect of biostimulants on 2,4,6-trinitrotoluene (TNT) degradation and bacterial community composition in contaminated aquifer sediment enrichments

2,4,6-Trinitrotoluene (TNT) is a toxic and persistent explosive compound occurring as a contaminant at numerous sites worldwide. Knowledge of the microbial dynamics driving TNT biodegradation is limited, particularly in native aquifer sediments where it poses a threat to water resources. The purpose of this study was to quantify the effect of organic amendments on anaerobic TNT biodegradation rate and pathway in an enrichment culture obtained from historically contaminated aquifer sediment and to compare the bacterial community dynamics. TNT readily biodegraded in all microcosms, with the highest biodegradation rate obtained under the lactate amended condition followed by ethanol amended and naturally occurring organic matter (extracted from site sediment) amended conditions. Although a reductive pathway of TNT degradation was observed across all conditions, denaturing gradient gel electrophoresis (DGGE) analysis revealed distinct bacterial community compositions. In all microcosms, Gram-negative γ- or β-Proteobacteria and Gram-positive Negativicutes or Clostridia were observed. A Pseudomonas sp. in particular was observed to be stimulated under all conditions. According to non-metric multidimensional scaling analysis of DGGE profiles, the microcosm communities were most similar to heavily TNT-contaminated field site sediment, relative to moderately and uncontaminated sediments, suggesting that TNT contamination itself is a major driver of microbial community structure. Overall these results provide a new line of evidence of the key bacteria driving TNT degradation in aquifer sediments and their dynamics in response to organic carbon amendment, supporting this approach as a promising technology for stimulating in situ TNT bioremediation in the subsurface.     

Anaerobic benzene degradation by bacteria

Benzene is a widespread and toxic contaminant. The fate of benzene in contaminated aquifers seems to be primarily controlled by the abundance of oxygen: benzene is aerobically degraded at high rates by ubiquitous microorganisms, and the oxygen‐dependent pathways for its breakdown were elucidated more than 50 years ago. In contrast, benzene was thought to be persistent under anoxic conditions until 25 years ago. Nevertheless, within the last 15 years, several benzene‐degrading cultures have been enriched under varying electron acceptor conditions in laboratories around the world, and organisms involved in anaerobic benzene degradation have been identified, indicating that anaerobic benzene degradation is a relevant environmental process. However, only a few benzene degraders have been isolated in pure culture so far, and they all use nitrate as an electron acceptor. In some highly enriched strictly anaerobic cultures, benzene has been described to be mineralized cooperatively by two or more different organisms. Despite great efforts, the biochemical mechanism by which the aromatic ring of benzene is activated in the absence of oxygen is still not fully elucidated; methylation, hydroxylation and carboxylation are discussed as likely reactions. This review summarizes the current knowledge about the ‘key players’ of anaerobic benzene degradation under different electron acceptor conditions and the possible pathway(s) of anaerobic benzene degradation.     

Anaerobic degradation of benzene in BTX mixtures dependent on sulfate reduction

Abstract Enrichment cultures from marine sediments mineralized benzene while using sulfate as the terminal electron acceptor. Parallel cultures using river marsh sediment displayed no activity. Mineralization was confirmed by release of ¹⁴CO₂ from radiolabeled benzene. The dependence on sulfate reduction was demonstrated by stoichiometric balances and the use of specific inhibitors. This work supports recent observations that anaerobic benzene degradation takes place coupled to sulfate reduction.     

Biodegradability of chlorinated solvents and related chlorinated aliphatic compounds

The biodegradability of chlorinated methanes, chlorinated ethanes, chlorinated ethenes, chlorofluorocarbons (CFCs), chlorinated acetic acids, chlorinated propanoids and chlorinated butadienes was evaluated based on literature data. Evidence for the biodegradation of compounds in all of the compound categories evaluated has been reported. A broad range of chlorinated aliphatic structures are susceptible to biodegradation under a variety of physiological and redox conditions. Microbial biodegradation of a wide variety of chlorinated aliphatic compounds was shown to occur under five physiological conditions. However, any given physiological condition could only act upon a subset of the chlorinated compounds. Firstly, chlorinated compounds are used as an electron donor and carbon source under aerobic conditions. Secondly, chlorinated compounds are cometabolized under aerobic conditions while the microorganisms are growing (or otherwise already have grown) on another primary substrate. Thirdly, chlorinated compounds are also degraded under anaerobic conditions in which they are utilized as an electron donor and carbon source. Fourthly, chlorinated compounds can serve as an electron acceptor to support respiration of anaerobic microorganisms utilizing simple electron donating substrates. Lastly chlorinated compounds are subject to anaerobic cometabolism becoming biotransformed while the microorganisms grow on other primary substrate or electron acceptor. The literature survey demonstrates that, in many cases, chlorinated compounds are completely mineralised to benign end products. Additionally, biodegradation can occur rapidly. Growth rates exceeding 1 d^-1 were observed for many compounds. Most compound categories include chlorinated structures that are used to support microbial growth. Growth can be due to the use of the chlorinated compound as an electron donor or alternatively to the use of the chlorinated compound as an electron acceptor (halorespiration). Biodegradation linked to growth is important, since under such conditions, rates of degradation will increase as the microbial population (biocatalyst) increases. Combinations of redox conditions are favorable for the biodegradation of highly chlorinated structures that are recalcitrant to degradation under aerobic conditions. However, under anaerobic conditions, highly chlorinated structures are partially dehalogenated to lower chlorinated counterparts. The lower chlorinated compounds are subsequently more readily mineralized under aerobic conditions.     

Bioremediation of Hexachlorocyclohexane Isomers,Chlorinated Benzenes,and Chlorinated Ethenes in Soil and Fractured Dolomite

Groundwater at an industrial site is contaminated with α hexachlorocyclohexane (HCH) and γ -HCH (i.e., lindane) (0.3 to 0.5 ppm). Other contaminants in the 1 to 15 ppm range include 1,2,4-trichlorobenezene (TCB), 1,2-dichlorobenzene (DCB), 1,3-DCB, 1,4-DCB, chlorobenzene (CB), benzene, trichloroethene (TCE), and cis-1,2-dichloroethene (cDCE). The aquifer consists of a shallow layer of soil over fractured dolomite, where most of the contaminant mass resides. The objective of this study was to compare (1) anaerobic reductive dechlorination of the polychlorinated contaminants, followed by aerobic biodegradation of the daughter products (mainly DCBs, CB, and benzene); and (2) aerobic biodegradation of α - and γ -HCH, TCB, DCBs, CB, and benzene, followed by anaerobic reduction of TCE and cDCE to ethene. Conventional wisdom suggests that sequential anaerobic and aerobic conditions are desirable for bioremediating sites contaminated by mixtures of polychlorinated organics. The results of this microcosm study suggest that a sequential aerobic and anaerobic approach may be more successful, although implementing this in the field presents some major challenges. In the dolomite microcosms incubated under aerobic conditions first (59 days), α - and γ -HCH were biodegraded close to the maximum contaminant level for lindane; all of the aromatic compounds were consumed; and there was partial removal of TCE and cDCE (presumptively via cometabolism). The subsequent switch to anaerobic conditions (day 101) yielded reductive dechlorination of the remaining TCE; a significant level of ethene was produced, although some cDCE and VC persisted. In contrast, sequential anaerobic (393 days) and aerobic treatment (498 days) for the dolomite microcosms was ineffective in completely removing the aromatic compounds, α -HCH, cDCE, and VC. For the soil microcosms, both treatment sequences were effective, most likely reflecting a greater abundance of the necessary microbes and electron donor in this part of the site.     

硝酸盐还原促进毒害性有机污染物降解的研究进展

大量具有高毒性、持久性和生物蓄积性的有机污染物被排放到环境中,对生态环境和人类健康造成了严重威胁。近年来,利用硝酸盐作电子受体在厌氧条件下降解毒害性有机污染物,已取得一定的进展。本文综述了硝酸盐还原体系中几种典型毒害性有机污染物(多环芳烃、单环或杂环芳烃类有机物及卤代有机物)的厌氧降解研究进展。在此基础上,提出了硝酸盐还原促进毒害性有机污染物降解研究中存在的主要问题及其在加速污染环境净化方面的应用前景。     

Anaerobic benzene degradation under denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence for a syntrophic process

An anaerobic microbial community was enriched in a chemostat that was operated for more than 8 years with benzene and nitrate as electron acceptor. The coexistence of multiple species in the chemostat and the presence of a biofilm, led to the hypothesis that benzene-degrading species coexist in a syntrophic interaction, and that benzene can be degraded in syntrophy by consortia with various electron acceptors in the same culture. The benzene-degrading microorganisms were identified by DNA-stable isotope probing with [U-(13) C]-labelled benzene, and the effect of different electron donors and acceptors on benzene degradation was investigated. The degradation rate constant of benzene with nitrate (0.7 day(-1) ) was higher than reported previously. In the absence of nitrate, the microbial community was able to use sulfate, chlorate or ferric iron as electron acceptor. Bacteria belonging to the Peptococcaceae were identified as dominant benzene consumers, but also those related to Rhodocyclaceae and Burkholderiaceae were found to be associated with the anaerobic benzene degradation process. The benzene degradation activity in the chemostat was associated with microbial growth in biofilms. This, together with the inhibiting effect of hydrogen and the ability to degrade benzene with different electron acceptors, suggests that benzene was degraded via a syntrophic process.