Anaerobic biotransformations of pollutant chemicals in aquifers

Summary Anaerobic microbial communities sampled from either a methanogenic or sulfate-reducing aquifer site have been tested for their ability to degrade a variety of groundwater pollutants, including halogenated aromatic compounds, simple alkyl phenols and tetrachloroethylene. The haloaromatic chemicals were biodegraded in methanogenic incubations but not under sulfate-reducing conditions. The primary degradative event was typically the reductive removal of the aryl halides. Complete dehalogenation of the aromatic moiety was required before substrate mineralization was observed. The lack of dehalogenation activity in sulfatereducing incubations was due, at least in part, to the high levels of sulfate rather than a lack of metabolic potential. In contrast, the degradation of cresol isomers occurred in both types of incubations but proved faster under sulfate-reducing conditions. The requisite microorganisms were enriched and the degradation pathway forp-cresol under the latter conditions involved the anaerobic oxidation of the aryl methyl group. Tetrachloroethylene was also degraded by reductive dehalogenation but under both incubation conditions. The initial conversion of this substrate to trichloroethylene was generally faster under methanogenic conditions. However, the transformation pathway slowed when dichloroethylene was produced and only trace concentrations of vinyl chloride were detected. These results illustrate that pollutant compounds can be biodegraded under anoxic conditions and a knowledge of the predominant ecological conditions is essential for accurate predictions of the transport and fate of such materials in aquifers.     

Biodegradation of aromatic compounds under mixed oxygen/denitrifying conditions: a review

Bioremediation of aromatic hydrocarbons in groundwater and sediments is often limited by dissolved oxygen. Many aromatic hydrocarbons degrade very slowly or not at all under anaerobic conditions. Nitrate is a good alternative electron acceptor to oxygen, and denitrifying bacteria are commonly found in the subsurface and in association with contaminated aquifer materials. Providing both nitrate and microaerophilic levels of oxygen may result in oxidation of the stable benzene rings in aromatic contaminants and allow for the intermediates of this oxidation to degrade via denitrification. The effects of using mixed electron acceptors on biodegradation of subsurface contaminants is unclear. Below some critical oxygen threshold, aerobic biodegradation is inhibited, however high levels of oxygen inhibit denitrification. The mechanisms which regulate electron transfer to oxygen and nitrate are complex. This review: 1) describes the factors which may affect the utilization of oxygen and nitrate as dual electron acceptors during biodegradation; 2) summarizes the incidence of dual use of nitrate and oxygen (aerobic denitrification); and 3) presents evidence of the effectiveness of bioremediation under mixed oxygen/nitrate conditions. Received 08 November 1995/ Accepted in revised form 09 June 1996     

Anaerobic biodegradation of no. 2 diesel fuel in soil: a soil column study

Soil and sediments are contaminated with petroleum hydrocarbons in many parts of the world. Anaerobic degradation of petroleum hydrocarbon is very relevant in removing oil spills in the anaerobic zones of soil and sediments. This research investigates the possibility of degrading no. diesel fuel under anaerobic conditions. Anaerobic packed soil columns were used to simulate and study in situ bioremediation of soil contaminated with diesel fuel. Several anaerobic conditions were evaluated in soil columns, including sulfate reducing, nitrate reducing, methanogenic, and mixed electron acceptor conditions. The objectives were to determine the extent of diesel fuel degradation in soil columns under various anaerobic conditions and identify the best conditions for efficient removal of diesel fuel. Diesel fuels were degraded significantly under all conditions compared to no electron supplemented soil column (natural attenuation). However, the rate of diesel degradation was the highest under mixed electron acceptor conditions followed in order by sulfate reducing, nitrate reducing, and methanogenic conditions. Under mixed electron acceptor condition 81% of diesel fuel was degraded within 310 days. While under sulfate reducing condition 54.5% degradation of diesel fuel was observed for the same period. This study showed evidence for diesel fuel metabolism in a mixed microbial population system similar to any contaminated field sites, where heterogeneous microbial population exists.     

On the relationship between methane production and oxidation by anaerobic methanotrophic communities from cold seeps of the Gulf of Mexico

The anaerobic oxidation of methane (AOM) in the marine subsurface is a significant sink for methane in the environment, yet our understanding of its regulation and dynamics is still incomplete. Relatively few groups of microorganisms consume methane in subsurface environments – namely the anaerobic methanotrophic archaea (ANME clades 1, 2 and 3), which are phylogenetically related to methanogenic archaea. Anaerobic oxidation of methane presumably proceeds via a 'reversed' methanogenic pathway. The ANME are generally associated with sulfate-reducing bacteria (SRB) and sulfate is the only documented final electron acceptor for AOM in marine sediments. Our comparative study explored the coupling of AOM with sulfate reduction (SR) and methane generation (MOG) in microbial communities from Gulf of Mexico cold seep sediments that were naturally enriched with methane and other hydrocarbons. These sediments harbour a variety of ANME clades and SRB. Following enrichment under an atmosphere of methane, AOM fuelled 50–100% of SR, even in sediment slurries containing petroleum-associated hydrocarbons and organic matter. In the presence of methane and sulfate, the investigated microbial communities produce methane at a small fraction (∼10%) of the AOM rate. Anaerobic oxidation of methane, MOG and SR rates decreased significantly with decreasing concentration of methane, and in the presence of the SR inhibitor molybdate, but reacted differently to the MOG inhibitor 2-bromoethanesulfonate (BES). The addition of acetate, a possible breakdown product of petroleum in situ and a potential intermediate in AOM/SR syntrophy, did not suppress AOM activity; rather acetate stimulated microbial activity in oily sediment slurries.     

Exocellular electron transfer in anaerobic microbial communities

Exocellular electron transfer plays an important role in anaerobic microbial communities that degrade organic matter. Interspecies hydrogen transfer between microorganisms is the driving force for complete biodegradation in methanogenic environments. Many organic compounds are degraded by obligatory syntrophic consortia of proton-reducing acetogenic bacteria and hydrogen-consuming methanogenic archaea. Anaerobic microorganisms that use insoluble electron acceptors for growth, such as iron- and manganese-oxide as well as inert graphite electrodes in microbial fuel cells, also transfer electrons exocellularly. Soluble compounds, like humic substances, quinones, phenazines and riboflavin, can function as exocellular electron mediators enhancing this type of anaerobic respiration. However, direct electron transfer by cell-cell contact is important as well. This review addresses the mechanisms of exocellular electron transfer in anaerobic microbial communities. There are fundamental differences but also similarities between electron transfer to another microorganism or to an insoluble electron acceptor. The physical separation of the electron donor and electron acceptor metabolism allows energy conservation in compounds as methane and hydrogen or as electricity. Furthermore, this separation is essential in the donation or acceptance of electrons in some environmental technological processes, e.g. soil remediation, wastewater purification and corrosion.     

Role of sulfate in microbial transformations of environmental contaminants: Chlorinated aromatic compounds

Despite recent progress made in describing microbial transformations that occur under anaerobic conditions, our understanding of the role sulfate‐reducing bacteria may play in the remediation of environmental contaminants is still very limited. The objective of this mini‐review is to summarize what is currently known of the metabolism of chlorinated aromatic compounds in the presence of sulfate. Sulfidogenic processes are discussed with respect to the thermodynamics of haloaromatic oxidation and to their potential use in the in situ bioremediation of hazardous organic wastes. A comprehensive listing is made of anaerobic transformations that involve both halogenated and nonhalogenated monoaromatic substrates by denitrifiers, dissimilatory iron‐reducing bacteria, and methanogenic consortia. In contrast to other anaerobic processes, studies involving sulfate‐mediated metabolism of hazardous organic compounds have been neglected; however, the recent success in defining methanogenic transformations, in particular, has enhanced expectations of defining an analogous role for sulfate‐reducing microbial communities in low redox environments that have become contaminated with hazardous substances.     

Reduction of Prussian Blue by the two iron-reducing microorganisms Geobacter metallireducens and Shewanella alga

Cyanide or cyanide-metal complexes are frequent contaminants of soil or aquifers at industrial sites, which can be released from such sites by outgassing or transport with the groundwater. They form very stable complexes with iron, which may occur in the subsurface as an insoluble blue mineral, the so-called Prussian Blue (Fe(4)[Fe(CN)(6)](3)). In this study, we show that the insoluble and colloidal Fe(III)-cyanide complex Prussian Blue can be reduced and utilized as electron acceptor by the dissimilatory iron-reducing bacteria Geobacter metallireducens and Shewanella alga strain BrY. The microbial reduction of the dark blue pigment Prussian Blue leads to the formation of a completely colourless solid mineral, presumably Prussian White (Fe(2)[Fe(CN)(6)]), which could be reoxidized through exposure to air, regaining the dark blue colour. In addition, the microorganisms were able to grow with Prussian Blue, using it as the sole electron acceptor. Geobacter metallireducens could also reduce Prussian Blue coatings on sand, which was sampled from a contaminated site.     

Microbial diversity of methanogenic communities in the systems for anaerobic treatment of organic waste

Methane production via anaerobic degradation of organic-contaminated wastewater, semiliquid, or solid municipal waste of complex composition by methanogenic microbial communities is a multistage process involving at least four groups of microorganisms. These are hydrolytic bacteria (polysaccharolytic, proteolytic, and lipolytic), fermentative bacteria, acetogenic bacteria (syntrophic, proton-reducing), and methanogenic archaea; complex trophic interactions exist between these groups. The review provides information concerning the diversity of the major microbial groups identified in the systems for wastewater and concentrated waste treatment, solid-phase anaerobic fermentation, and landfills for disposal of municipal solid waste, and also specifies the sources of isolation of the type strains. The research demonstrates that both new microorganisms and those previously isolated from natural habitats may be found in waste treatment systems. High microbial diversity in the systems for organic waste treatment provides for stable methanogenesis under fluctuating environmental conditions.     

Metabolic diversity of aromatic hydrocarbon-degrading bacteria from a petroleum-contaminated aquifer

We characterized bacteria from contaminated aquifers for their ability to utilize aromatic hydrocarbons under hypoxic (oxygen-limiting) conditions (initial dissolved oxygen concentration about 2 mg/l) with nitrate as an alternate electron acceptor. This is relevant to current intense efforts to establish favorable conditions forin situ bioremediation. Using samples of granular activated carbon slurries from an operating groundwater treatment system, we isolated bacteria that are able to use benzene, toluene, ethylbenzene, orp-xylene as their sole source of carbon under aerobic or hypoxic-denitrifying conditions. Direct isolation on solid medium incubated aerobically or hypoxically with the substrate supplied as vapor yielded 10³ to 10⁵ bacteria ml^–1 of slurry supernatant, with numbers varying little with respect to isolation substrate or conditions. More than sixty bacterial isolates that varied in colony morphology were purified and characterized according to substrate utilization profiles and growth condition (i.e., aerobic vs. hypoxic) specificity. Strains with distinct characteristics were obtained using benzene compared with those isolated on toluene or ethylbenzene. In general, isolates obtained from direct selection on benzene minimal medium grew well under aerobic conditions but poorly under hypoxic conditions, whereas many ethylbenzene isolates grew well under both incubation conditions. We conclude that the conditions of isolation, rather than the substrate used, will influence the apparent characteristic substrate utilization range of the isolates obtained. Also, using an enrichment culture technique, we isolated a strain ofPseudomonas fluorescens, designated CFS215, which exhibited nitrate dependent degradation of aromatic hydrocarbons under hypoxic conditions.Abbreviations BTEX benzene, toluene, ethylbenzene, andp-xylene - HPLC high performance liquid chromatography - GAC granular activated carbon     

Physiological and molecular characterization of a microbial community established in unsaturated, petroleum-contaminated soil

The microbial communities established in soil samples from an unsaturated, petroleum-contaminated zone and from an adjacent uncontaminated site were characterized by physiological and molecular approaches. Possible electron acceptors such as sulfate and nitrate had been completely depleted in these soil samples. Slurries of these soil samples were incubated in bottles in the presence of hydrocarbon indicators (benzene, toluene, xylene and decane), and the degradation of these compounds was examined. Supplementation with electron acceptors stimulated hydrocarbon degradation, although the stimulatory effect was small in the contaminated soil. The initial degradation rates in the contaminated soil under fermentative/methanogenic conditions were comparable to those under aerobic conditions. The microbial populations in the original soil samples were analysed by cloning and sequencing of polymerase chain reaction (PCR)-amplified bacterial and archaeal 16S rRNA gene fragments, showing that the sequences retrieved from these soils were substantially different. For instance, Epsilonproteobacteria, Gammaproteobacteria, Crenarchaeota and Methanosarcinales could only be detected at significant levels in the contaminated soil. Denaturing gradient gel electrophoresis (DGGE) analyses of 16S rRNA gene fragments amplified by PCR from the incubated soil-slurry samples showed that supplementation of the electron acceptors resulted in a shift in the major populations, while the DGGE profiles after incubating the contaminated soil under the fermentative/methanogenic conditions were not substantially changed. These results suggest that petroleum contamination of the unsaturated zone resulted in the establishment of a fermentative/methanogenic community with substantial hydrocarbon-degrading potential.     

Changes in bacterial communities from anaerobic digesters during petroleum hydrocarbon degradation

Anaerobic biodegradation of petroleum hydrocarbons (PHC) to methane has been recognized to occur in oil reservoirs and contaminated surface sites alike. This process could be employed efficiently for the treatment of contaminated materials, including petrochemical wastes and PHC-contaminated soil, since no external electron acceptor is required. Moreover, the controlled production of methane in digestion plants, similarly to the anaerobic digestion (AD) of energy crops or organic residues, would enable for energy recovery from these wastes. At present, little is known about the bacterial communities involved in and responsible for hydrocarbon fermentation, the initial step in PHC conversion to methane. In the present study, the fate of two different methanogenic communities derived from the AD of wastewater (WWT) and of biowaste, mixed with PHC-contaminated soil (SWT), was monitored during incubation with PHC using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA genes amplified with Bacteria-specific primers. During 11 months of incubation, slight but significant degradation of PHC occurred in both sludges and distinct bacterial communities were developing. In both sludges, Bacteroidetes were found. In addition, in WWT, the bacterial community was found to be dominated by Synergistetes and Proteobacteria, while Firmicutes and unidentified members were abundant in SWT. These results indicate that bacterial communities from anaerobic digesters can adapt to and degrade petroleum hydrocarbons. The decontamination of PHC-containing waste via fermentative treatment appears possible.     

Bacterial Diversity and Biogeochemical Processes of Oil-Contaminated Groundwater,Baoding, North China

A total of 43 groundwater samples were collected from 9 multimonitoring wells at a petrochemical site, Baoding City, North China, from June 2008 to December 2009 to investigate the biogeochemical processes and/or bacterial conmmunity using both culture-dependent and -independent methods. The results showed that aromatic hydrocarbons and chlorinated hydrocarbons were the major pollutants in the groundwater. Denitrification and iron reduction might be the main biogeochemical processes in the aquifers at this site, which seemed to transform from denitrification-dominated to iron reduction-dominated in some sections. Denaturing gradient gel electrophoresis (DGGE) revealed that the dominant bacterial groups of the groundwater were related to some oil-degrading bacteria, which can grow under denitrifying, iron-reducing and sulfate-reducing anaerobic conditions. In some serious contaminated groundwater niches, there might be sulfur cycles, as sulfur oxidizer was also abundant, which was further confirmed by 16S rRNA gene cloning analysis. The operational taxonomic units (OTUs) that highly related to Pseudomonas sp., Hydrogenophaga sp., Sphingomonas sp., Ferribacterium sp. and Sulfuricurvum Kujiense etc. were predominant in the groundwater contaminated by chlorinated hydrocarbons (CHCs), benzene, toluene, ethylbenzene, and xylenes (BTEX) and/or polycyclic aromatic hydrocarbons (PAHs), respectively. Biodiversity seemed to be undermined by oil contamination, and varied with seasons. The bacterial community in the contaminated groundwater was largely determined by the groundwater geochemistry.     

Assessment of microbial communities associated with fermentative–methanogenic biodegradation of aromatic hydrocarbons in groundwater contaminated with a biodiesel blend (B20)

A controlled field experiment was conducted to assess the potential for fermentative–methanogenic biostimulation (by ammonium-acetate injection) to enhance biodegradation of benzene, toluene, ethylbenzene and xylenes (BTEX) as well as polycyclic aromatic hydrocarbons (PAHs) in groundwater contaminated with biodiesel B20 (20:80 v/v soybean biodiesel and diesel). Changes in microbial community structure were assessed by pyrosequencing 16S rRNA analyses. BTEX and PAH removal began 0.7 year following the release, concomitantly with the increase in the relative abundance of Desulfitobacterium and Geobacter spp. (from 5 to 52.7 % and 15.8 to 37.3 % of total Bacteria 16S rRNA, respectively), which are known to anaerobically degrade hydrocarbons. The accumulation of anaerobic metabolites acetate and hydrogen that could hinder the thermodynamic feasibility of BTEX and PAH biotransformations under fermentative/methanogenic conditions was apparently alleviated by the growing predominance of Methanosarcina. This suggests the importance of microbial population shifts that enrich microorganisms capable of interacting syntrophically to enhance the feasibility of fermentative–methanogenic bioremediation of biodiesel blend releases.     

Naphthalene and Benzene Degradation under Fe(III)-Reducing Conditions in Petroleum-Contaminated Aquifers

Naphthalene was oxidized anaerobically to CO₂ in sediments collected from a petroleum-contaminated aquifer in Bemidji, Minnesota in which Fe(III) reduction was the terminal electron-accepting process. Naphthalene was not oxidized in sediments from the methanogenic zone at Bemidji or in sediments from the Fe(III)-reducing zone of other petroleum-contaminated aquifers studied. In a profile across the Fe(III)-reducing zone of the Bemidji aquifer, rates of naphthalene oxidation were fastest in sediments with the highest proportion of Fe(III), which was also the zone of the most rapid degradation of benzene, toluene, and acetate. The comparative studies attempted to elucidate factors that might account for the fact that unsubstituted aromatic hydrocarbons such as benzene and naphthalene were degraded under Fe(III)-reducing conditions at Bemidji, but not at the other aquifers examined. These studies indicated that the ability of Fe(III)-reducing microorganisms to degrade benzene and naphthalene at the Bemidji site cannot be attributed to groundwater components that make Fe(III) more available for reduction or other potential factors that were evaluated. However, unlike the other aquifers evaluated, uncontaminated sediments at the Bemidji site could be adapted for anaerobic benzene degradation merely with the addition of benzene. These findings indicate that Bemidji sediments naturally contain Fe(III) reducers capable of degradation of unsubstituted aromatic hydrocarbons.     

In situ bacterial colonization of compacted bentonite under deep geological high-level radioactive waste repository conditions

Subsurface microorganisms are expected to invade, colonize, and influence the safety performance of deep geological spent nuclear fuel (SNF) repositories. An understanding of the interactions of subsurface dwelling microbial communities with the storage is thus essential. For this to be achieved, experiments must be conducted under in situ conditions. We investigated the presence of groundwater microorganisms in repository bentonite saturated with groundwater recovered from tests conducted at the Äspö underground Hard Rock Laboratory in Sweden. A 16S ribosomal RNA and dissimilatory bisulfite reductase gene distribution between the bentonite and groundwater samples suggested that the sulfate-reducing bacteria widespread in the aquifers were not common in the clay. Aerophilic bacteria could be cultured from samples run at ≤55°C but not at ≥67°C. Generally, the largely gram-negative groundwater microorganisms were poorly represented in the bentonite while the gram-positive bacteria commonly found in the clay predominated. Thus, bentonite compacted to a density of approximately 2 g cm^?3 together with elevated temperatures might discourage the mass introduction of the predominantly mesophilic granitic aquifer bacteria into future SNF repositories in the long run.     

Anaerobic degradation of alkylated benzenes in denitrifying laboratory aquifer columns

Toluene and m-xylene were rapidly mineralized in an anaerobic laboratory aquifer column operated under continuous-flow conditions with nitrate as an electron acceptor. The oxidation of toluene and m-xylene was coupled with the reduction of nitrate, and mineralization was confirmed by trapping 14CO2 evolved from 14C-ring-labeled substrates. Substrate degradation also took place when nitrous oxide replaced nitrate as an electron acceptor, but decomposition was inhibited in the presence of molecular oxygen or after the substitution of nitrate by nitrite. The m-xylene-adapted microorganisms in the aquifer column degraded toluene, benzaldehyde, benzoate, m-toluylaldehyde, m-toluate, m-cresol, p-cresol, and p-hydroxybenzoate but were unable to metabolize benzene, naphthalene, methylcyclohexane, and 1,3-dimethylcyclohexane. Isotope-dilution experiments suggested benzoate as an intermediate formed during anaerobic toluene metabolism. The finding that the highly water-soluble nitrous oxide served as electron acceptor for the anaerobic mineralization of some aromatic hydrocarbons may offer attractive options for the in situ restoration of polluted aquifers.     

Anaerobic degradation of alkylated benzenes in denitrifying laboratory aquifer columns.

Toluene and m-xylene were rapidly mineralized in an anaerobic laboratory aquifer column operated under continuous-flow conditions with nitrate as an electron acceptor. The oxidation of toluene and m-xylene was coupled with the reduction of nitrate, and mineralization was confirmed by trapping 14CO2 evolved from 14C-ring-labeled substrates. Substrate degradation also took place when nitrous oxide replaced nitrate as an electron acceptor, but decomposition was inhibited in the presence of molecular oxygen or after the substitution of nitrate by nitrite. The m-xylene-adapted microorganisms in the aquifer column degraded toluene, benzaldehyde, benzoate, m-toluylaldehyde, m-toluate, m-cresol, p-cresol, and p-hydroxybenzoate but were unable to metabolize benzene, naphthalene, methylcyclohexane, and 1,3-dimethylcyclohexane. Isotope-dilution experiments suggested benzoate as an intermediate formed during anaerobic toluene metabolism. The finding that the highly water-soluble nitrous oxide served as electron acceptor for the anaerobic mineralization of some aromatic hydrocarbons may offer attractive options for the in situ restoration of polluted aquifers.     

Enhanced Biotransformation of Trichloroethylene Under Mixed Electron Acceptor Conditions

The biotransformation of trichloroethylene (TCE) under various electron acceptor conditions was investigated by using enrichment cultures developed from the anaerobic digester sludge of Thibodaux sewage treatment plant. The results indicated that TCE was biotransformed under sulfate reducing, methanogenic, nitrate reducing, iron reducing, and fermenting conditions. However, the rates of TCE removal varied among the conditions studied. The fastest removal of TCE (100% removal in 9 days) was observed under mixed electron acceptor conditions, followed in order by methanogenic, fermenting, iron reducing, sulfate reducing, and nitrate reducing conditions. Under mixed electron acceptor conditions, the TCE was converted to ethene, which was further metabolized. Under sulfate and nitrate reducing conditions, the major metabolites produced from TCE metabolism were cis and trans dichloroethylene (DCE). Under methanogenic, iron reducing, and fermenting conditions, cis and trans DCE and ethene were produced from TCE metabolism. This study showed evidence for TCE metabolism in a mixed microbial population system similar to any contaminated field sites, where heterogeneous microbial population exists. Received: 21 July 2000 / Accepted: 5 September 2000     

O-Demethylation, dehydroxylation, ring-reduction and cleavage of aromatic substrates by Enterobacteriaceae under anaerobic conditions

Four fermentative facultative anaerobes, members of the genera Enterobacter and Escherichia , were tested for their ability to transform an aromatic lignin derivative, 3-methoxy-4-hydroxy-cinnamic acid (ferulic acid), under anaerobic (fermentative) conditions. The pure cultures studied were shown to O-demethylate, dehydroxylate, reduce the double bond in the side-chain, decarboxylate the aromatic ring to the stage of benzoate and to reduce the ring to an alicyclic acid. Aromatic hydrocarbons (toluene, ethylbenzene and propylbenzene), as well as phenols (phenol, o-cresol, p-cresol, 2-ethylphenol and 3-hydroxy-4-ethylphenol) were also produced. In addition, during 3 months incubation, the cleavage of the aromatic ring occurred, whereby a small fraction of the substrate was converted to straight-chain and branched (methylated, ethylated) five- to eight-carbon aliphatic acids. The results indicate that pure cultures of fermentative facultative anaerobes might be capable of degrading substituted aromatic acids to aliphatic products under strictly anaerobic (fermentative) conditions. These abilities, which have so far been found only in denitrifying pseudomonads among facultative anaerobes, might be common in Enterobacteriaceae. It is conceivable that these bacteria are important as degraders of aromatic compounds in anaerobic ecosystems.     

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.