Ann Microbiol (2015) 65:1201–1213 DOI 10.1007/s13213-014-0926-8

REVIEW ARTICLE

Microorganisms involved in anaerobic benzene degradation

Arturo Aburto-Medina & Andrew S. Ball

Received: 17 March 2014 /Accepted: 29 May 2014 /Published online: 26 June 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract Benzene is a highly toxic compound. Moreover, Keywords Benzene . Benzene-contaminated groundwater . benzene-contaminated groundwater is a widespread problem Benzene-degrading microorganisms . Syntrophic interactions caused mainly by the lack of oxygen in the subsurface. Long- term exposure may cause leukaemia. However, pure cultures of microorganisms with the ability to degrade benzene anaer- Introduction obically have recently been isolated from novel enrichment cultures. The novel pure cultures include the hyperthermo- Benzene is derived from fossil fuels, cigarette smoke and philic archaeon Ferroglobus placidus and the Geobacter forest fires, among other sources. It is an important raw strains Ben and metallireducens which degrade benzene an- chemical used as an intermediate to produce a wide range of aerobically coupled to the reduction of Fe(III). Syntrophic products, such as plastics, resins, nylon, lubricants and pesti- interactions have been suggested in enrichments where mem- cides. Benzene is also highly toxic; it may cause acute mye- bers of the Peptococcaceae within the class Clostridia are the loid leukaemia, secondary aplastic anaemia and damage to the dominant organisms and suggested as being responsible for reproductive system. It can enter the body by skin contact, the first attack on the benzene ring. Laboratory enrichment inhalation or consumption of contaminated water. Benzene- studies have also resulted in the development of consortia contaminated groundwater is a widespread problem caused which degrade benzene, with different terminal electron ac- partially by its high solubility in water (1.8 g l−1 at 25°C) ceptors supporting the syntrophy. Other benzene-degrading relative to other organic compounds and partly to spills at microorganisms have been identified under methanogenic production sites and leaks in underground storage tanks. The- conditions, involving the reduction of humic acids by, for se leaks have been documented by studies of the environment example, the deltaproteobacterium Hasda-A and members of agencies in the USA and UK (Rudolph 1996;Atlas2005). the Betaproteobacteria, Gammaproteobacteria and Benzene is readily degraded under aerobic conditions Deltaproteobacteria, respectively. This review focuses on (Alvarez and Vogel 1991; Werlen et al. 1996; Fairlee et al. the microorganisms involved in anaerobic benzene degrada- 1997; Greene et al. 2000) by ubiquitous microorganisms, such tion under conditions involving several electron acceptors in as Pseudomonas sp. and Rhodococcus sp. (Fahy et al. 2006). recent years. Information related to the anaerobic degradation However contaminated groundwater is usually under anaero- of benzene is critical to understanding and predicting the fate bic conditions due to the rapid depletion of available oxygen of this contaminant in groundwater. in subterranean environments, which in turn decreases the redox potential, favouring the growth of denitrifying, sulphate-reducing, iron-reducing and methanogenic popula- * A. Aburto-Medina ( ) tions that may also degrade the contaminant. Therefore, in- Department of Environmental Sciences, Universidad Autónoma Metropolitana, Lerma 52006, Mexico formed knowledge of anaerobic microorganisms capable of e-mail: [email protected] benzene degradation is critical to understanding and predicting the fate of this contaminant. A. S. Ball Initial studies suggested the lack of benzene degradation School of Applied Sciences, Centre for Environment, Sustainability and Remediation, Royal Melbourne Institute of Technology, under anaerobic conditions (Barker et al. 1987;Kuhnetal. Bundoora, VIC 3083, Australia 1988; Acton and Barker 1992; Barbaro et al. 1992; Patterson 1202 Ann Microbiol (2015) 65:1201–1213 et al. 1993). However, laboratory-based and field studies in 2001). However, no anaerobic degradation genes were found the last 20 years have confirmed the degradation of this in the Dechloromonas aromatica RCB genome (Salinero et al. pollutant coupled to the reduction of nitrate (Burland and 2009), and subsequent data suggests the involvement of oxy- Edwards 1999;Coatesetal.2001; Chakraborty and Coates gen under nitrate-reducing conditions (Ettwig et al. 2010; 2005; Ulrich et al. 2005; Kasai et al. 2006;Jakoncicetal. Zhang et al. 2013). Nevertheless, recent studies have reported 2007), sulphate (Lovley et al. 1995; Phelps et al. 1996; Weiner the novel pure cultures archaeon Ferroglobus placidus and Lovley 1998a; Sublette et al. 2006; Musat and Widdel (Holmes et al. 2011)andaGeobacter metallireducens strain 2008; Berlendis et al. 2010), iron (Lovley and Woodward named Ben (Zhang et al. 2012), both of which are capable of 1996;Kazumietal.1997; Rooney-Varga et al. 1999;Jahn anaerobic benzene degradation. The hyperthermophilic et al. 2005; Kunapuli et al. 2007), manganese (Villatoro- archaeon F. placidus is able to degrade benzene anaerobically Monzón et al. 2003, 2008), humic acids (Cervantes et al. under iron-reducing conditions, producing benzoate as the 2011) and methanogenesis (Vogel and Grbicgalic 1986; main metabolite, suggesting the carboxylation of benzene to Grbicgalic and Vogel 1987;Kazumietal.1997; Weiner and form benzoate (Holmes et al. 2011). The Geobacter strain Lovley 1998b; Caldwell and Suflita 2000;Ulrichand Ben, isolated from sediments within the ferric ion [Fe(III)]- Edwards 2003; Chang et al. 2005; Da Silva and Alvarez reducing zone in a petroleum-contaminated aquifer in 2007; Sakai et al. 2009). Bemidji, USA, was found to degrade benzene with ferric iron Moreover, recent studies on the novel pure cultures of and anthraquinone-2,6-disulfonate (AQDS) as the only elec- Geobacter species and the archaeon Ferroglobus placidus tron acceptors. In addition, G. metallireducens is also able to have confirmed the degradation of benzene coupled to iron- degrade benzene coupled to the reduction of Fe(III); trace reducing conditions and have also provided information on amounts of phenol were detected during the degradation of the metabolites produced during growth on benzene (Holmes benzene, suggesting phenol as an intermediate. This finding is et al. 2011; Zhang et al. 2012). In addition, syntrophic asso- also supported by the inhibition of benzene degradation by the ciations are also suggested in enrichments capable of benzene deletion of genes involved in the metabolism of phenol and by degradation where members of the Peptococcaceae are the their upregulation during benzene oxidation (Zhang et al. key players (Taubert et al. 2012; van der Zaan et al. 2012). 2012; Zhang et al. 2013). Other microorganisms involved in the degradation of benzene under methanogenic conditions or coupled to the reduction of humic acids include the deltaproteobacterium Hasda-A and Microorganisms identified in recent anaerobic benzene two phylotypes of Gammaproteobacteria, respectively degradation studies (Cervantes et al. 2011;Masumotoetal.2012). In this review, we focus on the microorganisms which have been identified in Genera of the family Peptococcaceae (phylum anaerobic benzene degradation studies in recent years and the Firmicutes,classClostridia) have been identified as the possible terminal electron acceptors. It should be noted that main benzene degraders with most of the terminal elec- excellent reviews on the anaerobic degradation of hydrocar- tron acceptors [sulphate, nitrate, Fe(III), chlorate], and bons (Boll et al. 2002; Foght 2008; Fuchs et al. 2011; syntrophic interactions are strongly suggested. The most Meckenstock and Mouttaki 2011;Flanaganetal.2013; recent study by the Vogt group involved a sulphate- Heider and Schühle 2013;Bolletal.2014), anaerobic micro- reducing enrichment analysed by the protein-SIP tech- bial communities in hydrocarbon-contaminated aquifers nique, with their observations indicating syntrophy be- (Kleinsteuber et al. 2012), the use of stable isotope probing tween a benzene-fermenting clostridial group, a (SIP) to identify anaerobic benzene and toluene degraders sulphate-reducing deltaproteobacterial group that uses (Cupples 2011) and the degradation of benzene under anaer- the metabolites generated during benzene fermentation obic conditions (Lovley 2000; Coates et al. 2002; Weelink and a putative scavenger group from Bacteroidetes/ et al. 2010;Vogtetal.2011) have been published during the Chlorobi (Taubert et al. 2012). This result is consistent past 12 years. with that of a previous study on the same consortium where acetate and hydrogen were strongly suggested as intermediate metabolites in the anaerobic benzene degra- Pure cultures capable of anaerobic benzene degradation dation process (Rakoczy et al. 2011). Members of the Cryptanaerobacter/Pelotomaculum group are suggested Until a few years ago there were only a few pure cultures able to be the benzene-fermenting group, as has been indicated to degrade benzene anaerobically, and all of these were in other studies from the same group (Kleinsteuber et al. coupled to nitrate reduction: an Azoarcus strain DN11 and 2008;Herrmannetal.2010). Moreover, the syntrophic AN9 (Kasai et al. 2006), a Bacillus cereus strain (Dou et al. interactions and the presence of the Peptococcaceae mem- 2010)andDechloromonas strains JJ and RCB (Coates et al. bers are also consistent with other enrichments. Van der Ann Microbiol (2015) 65:1201–1213 1203

Zaan and colleagues (2012) identified members of the Microorganisms identified in nitrate-reducing conditions Rhodocyclaceae, Burkholderiaceae but mainly the Peptococcaceae as benzene degraders in an enrichment Although benzene was thought to be recalcitrant, by the late capable of benzene degradation with several electron 1990s an enrichment culture established using a groundwater acceptors [nitrate, sulphate, Fe(III), chlorate]; degradation and soil inoculum demonstrated benzene degradation with was inhibited by the injection of hydrogen. Kunapuli and nitrate as the only electron acceptor. Nitrate was reduced as colleagues suggested syntrophy in an iron-reducing en- nitrite accumulated stochiometrically, and over 90 % of la- richment derived from a former Polish coal gasification belled benzene was recovered in the form of CO2,thereby site where members of the Peptococcaceae, only distant- confirming benzene degradation coupled to nitrate reduction. ly related to the cultured Thermincola, were suggested to However, benzene degraders were not identified (Burland and be the main benzene oxidizers. Other microorganisms Edwards 1999). identified included uncultured Deltaproteobacteria and Nevertheless, two pure culture strains capable of benzene members of the Actinobacteria (Kunapuli et al. 2007). oxidation coupled to the reduction of nitrate were later report- Later, anaerobic benzene degradation coupled to sulphate ed (Coates et al. 2001). The organisms were isolated from two reduction was reported for the same enrichment in the different environments in the USA: strain JJ was isolated from Meckenstock group (Abu Laban et al. 2009), and the Campus Lake sediments from the Southern Illinois Universi- enzymes involved in iron-reducing conditions were ty; strain RCB was isolated from Potomac River sediments. revealed (Abu Laban et al. 2010). Another enrichment Both strains were assigned to the genus Dechloromonas with- from an underground gas storage aquifer in France revealed in the Betaproteobacteria, and they were reported to oxidize a phylotypes related to the Pelobacter (Deltaproteobacteria), wide range of compounds (formate, acetate, propionate, bu- Thermotogales, Methanolobus and species not previously tyrate, lactate, succinate, pyruvate, benzoate, toluene) coupled involved in BTEX (benzene, toluene, ethylbenzene, to nitrate reduction. In a subsequent experiment using strain xylenes) degradation (Berlendis et al. 2010). Moreover, RCB, Chakraborty et al. (2005) reported the recovery of CO2 members of the Desulfobacteraceae family have also been from labelled benzene, which supported the degradation of reported to be the dominant organisms in sulphate- benzene by strain RCB in anoxic aquatic sediments. More- reducing and methanogenic enrichments (Musat and over, these authors found that the remaining BTEX com- Widdel 2008; Oka et al. 2008). Other Deltaproteobacteria pounds were also degraded by this strain. reported under methanogenic conditions include Synthrophus Two other pure cultures (DN11 and AN9) capable of gentianae (Sakai et al. 2009), which is consistent with previ- benzene degradation were isolated from gasoline- ous results (Dojka et al. 1998;Aburtoetal.2009). contaminated groundwater; these belong to the Azoarcus fam- Recent studies have identified a deltaproteobacterium, ily, also found within the Betaproteobacteria (Kasai et al. Hasda-A, which was isolated from non-contaminated lotus 2006). The isolation of these two pure cultures is consistent field soil in Japan as one of the main benzene degraders under with the identification of a dominant microbial population methanogenic conditions (Masumoto et al. 2012). Members closely related to Azoarcus species in a benzene-degrading, of the Comamonadaceae were isolated from benzene- denitrifying enrichment (Ulrich and Edwards 2003). contaminated groundwater in Canada where benzene degra- In 2010, Dou et al. (2010) reported the bacteria Bacillus dation coupled to nitrate and sulphate reduction was enhanced cereus to be another pure culture capable of anaerobic benzene by the addition of non-activated persulphate (Xiong et al. degradation under nitrate-reducing conditions. The strain, 2012). which was isolated from gasoline-contaminated soil in China, Two Gammaproteobacteria phylotypes have also was able to degrade benzene concentrations up to 150 mg l−1, been reported to be the dominant organisms in a producing phenol and benzoate as metabolites. However, as benzene-degrading enrichment from Mexico that used the anaerobic degradation of benzene coupled to nitrate re- AQDS (humic acid) as the terminal electron acceptor duction seems to involve the presence of molecular oxygen (Cervantes et al. 2011). Anaerobic benzene degradation generated intracellularly, the cultures isolated by these authors was also observed in salt marsh sediment from the may not be considered as true anaerobic degraders (Salinero Louisiana coast after the 2010 BP oil spill, but no et al. 2009;Vogtetal.2011). microorganisms were identified (Yu et al. 2012). Table 1 A recent study by van der Zaan and colleagues (2012) summarizes the microorganisms identified in successful confirmed the anaerobic degradation of benzene under anaerobic benzene degradation during the last years, and denitrifying conditions by bacteria belonging to the Fig. 1 shows their phylogenetic distribution. The degra- Rhodocyclaceae,theBurkholderiaceae and the dation of benzene with the different electron acceptors Peptococcaceae, the latter being the dominant group. How- and the microbial communities detected are discussed in ever, degradation was suggested to occur via a syntrophic greater detail in the following sections. process since sulphate, chlorate and Fe(III) iron were also 1204

Table 1 Microorganisms identified in successful anaerobic benzene degradation studies during the last five years

Source Terminal electron acceptor Dominant phylotypes Intermediates/ Benzene degradation rate Reference Suggested pathway

− Lovley group collection. Fe(III) Geobacter metallireducens (pure culture) Phenol/hydroxylation 3.66 μMday1 Zhang et al. 2013 − Petroleum-contaminated aquifer Fe(III), AQDS Geobacter strain Ben (pure culture) ND Fe(III): 3.42 μMday1 AQDS: Zhang et al. 2012 − Bemidji, MN, USA 2.51 μMday1 2− Benzene-contaminated aquifer SO4 Syntrophy Pelotomaculum/Cryptanaerobacter Hydrogen and acetate Taubert et al. 2012 Zeitz, Germany (Peptococcaceae) Desulfobacca (Syntrophaceae) Rakoczy et al. 2011 − Rhodothermus 3.6–5.7 μMday1 Herrmann et al. 2010 Epsilonproteobacteria Desulfovibrio 2− −1 Chemostat enrichment NO3,SO4 Fe(III), Peptococcaceae Hydrogen K max :0.7day (Highest reported) van der Zaan et al. 2012 chlorate Syntrophy Rhodocyclaceae Burkholderiaceae 2− Benzene-contaminated NO3 O4 Comamonadaceae ND 75.9–92.8 % degradation rate. Xiong et al. 2012 groundwater, Canada Algoriphagus spp. Firmicutes Treponema Desulfovibrio −1 −1 Non-contaminated lotus field CH4 Deltaproteobacterium Hasda-A ND 3.39 μMday ;0.51μMday Masumoto et al. 2012; soil,Tsuchiura, Japan Syntrophy Firmicutes Sakaietal.2009

DSMZ strain Fe(III) Ferroglobus placidus Benzoate/ 81.2 % of CO2 recovered from Holmes et al. 2011 carboxylation benzene in 30 days. − Sediment and soil enrichments, AQDS, HPSHA ML sediment: Desulfobacca acetoxidans, ND ML: 0.115 μMday1 PR: Cervantes et al. 2011 − Mexico Shewanella, Chloroflexi 0.017 μMday1 PR: Phylum TM-7, Pseudomonadaceae

Coal gasification site, Poland Fe(III) Carboxylation ND Abu Laban et al. 2010 65:1201 (2015) Microbiol Ann 2− −1 Deep aquifer, France SO4 Pelobacter ND 0.066 ppm day Berlendis et al. 2010

Fe(III), Ferric ion; DSMZ, German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen); AQDS, anthraquinone-2,6-disulfonate; HPSHA, highly purified soil humic acids; ND not determined; PR, hydrocarbon-contaminated soil; ML, hydrocarbon-contaminated sediment – 1213 Ann Microbiol (2015) 65:1201–1213 1205

Fig. 1 Distribution of anaerobic benzene-degrading microorganisms based on 16S rRNA sequence comparisons. One representative organism from each order of each phylum was selected based on current bacterial taxonomy available from the online taxonomic outlines of prokaryotes available online (NCBI 2013), Jukes and Cantor distance, neighbour-joining method. Names in bold represent those orders from which organisms have been observed in anaerobic benzene degradation studies. Names in bold and italics represent orders from which anaerobic benzene degraders have been isolated. Archeoglobales belonging in the Archaea were included as the outgroup. 0.01 Changes per nucleotide position

used as electron acceptors, and the degradation was inhibited presence of adapted microorganisms in situ. The microorgan- in the presence of hydrogen. In this study, t he degradation rate isms were later identified as members of the Geobacteriaceae. constant of benzene with nitrate was the highest among the A further study confirmed Geobacter to be the dominant electron acceptors and also higher than previously reported organism in an enrichment prepared with the benzene- (van der Zaan et al. 2012). A later study reported the enhance- degrading sediments (Rooney-Varga et al. 1999). The Lovley ment of benzene degradation under nitrate- and sulphate- group has studied in detail the degradation of benzene under reducing conditions by the addition of non-activated iron-reducing conditions and found that it can be enhanced by persulphate, which breaks down triethyl phosphate into the addition of Fe(III) chelators, such as nitrilotriacetic acid ortophosphate and promotes nitrate and sulphate utilization (Lovley et al. 1994). Insoluble Fe(III) oxides are solubilized (Xiong et al. 2012). The dominant microorganisms belonged by nitrilotriacetic acid, making them more accessible to to the Comamonadaceae family of the Betaproteobacteria Fe(III)-reducing microorganisms, thereby enhancing benzene and the Algoriphagus genus within the Bacteroidetes phylum, degradation (Lovley and Woodward 1996). Other iron chela- and nitrate and sulphite reductases have been reported for this tors, such as EDTA, ethanol diglycine, phosphates and humic genus (Yoon et al. 2005). acids, have also been found to stimulate the oxidation of benzene coupled to Fe(III) reduction in sediments from a petroleum-contaminated aquifer (Lovley et al. 1996b). The Microorganisms identified in iron-reducing conditions greatest degradation enhancement was observed with humic substances since they serve as intermediates in the reduction; Members of the Geobacteraceae within the the microorganisms transfer electrons to quinone moieties in Deltaproteobacteria are usually responsible for the degrada- the humic substances that are further transported to Fe(III) tion of BTEX coupled to iron reduction. An early study by oxides, reducing them; this process regenerates the quinone Anderson and colleagues used sediments from the Fe(III)- moieties that can again receive electrons from Fe(III)-reducing reducing zone of a petroleum-contaminated aquifer in microorganisms (Lovley et al. 1996a). Thus, multiple cycles Bemidji, Minnesota to prepare microcosms spiked with ben- of reduction and oxidation of humic substances can occur, and zene (Anderson et al. 1998). These authors did not observe thereductionofironcanoccurevenatlowhumicacid any lag period during the degradation, which suggested the concentrations. There is no need for contact between the 1206 Ann Microbiol (2015) 65:1201–1213 microorganisms and the Fe(III) oxide because humic sub- contains pathways for the anaerobic degradation of benzoate, stances accelerate the reduction of Fe(III); moreover, the latter toluene, phenol and p-cresol. Moreover, Geobacter they can reach Fe(III) oxides that the microorganisms can not metallireducens was also capable of benzene degradation and they react with more types of Fe(III). coupled to iron reduction, confirming the suggested role of Kunapuli and colleagues used SIP and a clone library to Geobacter species as hydrocarbon degraders in contaminated identify the microorganisms in a benzene-degrading enrich- environments (Zhang et al. 2012). A further study by the same ment coupled to iron-reducing conditions in soils from a group on the G. metallireducens strain strongly suggested the former Polish coal gasification site (Kunapuli et al. 2007). presence of phenol as an intermediate since the genes coding The clones were related to the Peptococcaceae within the for enzyme subunits for the first and second steps in phenol Clostridia (BF1 cluster) and the Desulfobulbaceae within metabolism, PpsA and PpcB, respectively, were upregulated the Deltaproteobacteria. The closest cultured relatives of the during growth on benzene and their deletion inhibited benzene Peptococcaceae-related clones were Thermincola degradation (Zhang et al. 2013). Moreover 18O-labelled carboxydiphila and T. ferriacetica, while the phenol was recovered from labelled water, suggesting that Desulfobulbaceae-related clones were similar to other clones the phenol hydroxyl group was derived from water, as found in contaminated aquifers and in situ reactor clones previously suggested by Vogel and Grbicgalic (1986), but monitoring monochlorobenzene degradation (Alfreider et al. for methanogenic cultures. Thus, these results provide evi- 2002). A syntrophic mechanism was also proposed where the dence that phenol is a key intermediate in the degradation Peptococcaceae members were responsible for the first attack pathway and support hydroxylation as the first step in the on the benzene ring, while the Desulfobulbaceae members anaerobic degradation of benzene by G. metallireducens consumed the electrons released by benzene and assimilated (Zhang et al. 2013). carbon from the media. Acetate or other fermentation products were discarded as potential metabolites since they were not labelled, and only iron could be used as the terminal electron Microorganisms identified in sulphate-reducing acceptor. conditions A following study from the same group identified the enzymes responsible for anaerobic benzene degradation in The degradation of benzene coupled to sulphate reduction has the iron-reducing enrichment culture (Abu Laban et al. been documented and the main microorganisms identified; 2010). Proteins similar to the phenylphosphate carboxylase however, no sulphate-reducing pure culture has been isolated PpcA and PpcD of Azoarcus strain EbN1 and to the benzoate- (Edwards and Grbicgalic 1992; Chaudhuri and Wiesmann CoA ligase of Geobacter metallireducens were expressed 1995; Lovley et al. 1995; Phelps et al. 1998; Weiner et al. during benzene degradation, which suggested a direct carbox- 1998; Weiner and Lovley 1998a;Giegetal.1999; Sublette ylation of the benzene ring by a putative anaerobic benzene et al. 2006). carboxylase. Benzene degradation under iron-reducing condi- The first study to suggest the anaerobic oxidation of ben- tions has also been observed in salt marsh sediments, but the zene under sulphate-reducing conditions involved enrichment microorganisms were not identified (Yu et al. 2012). To date, cultures established with aquifer sediments from Seal Beach the hyperthermophilic Ferroglobus placidus is the only California and amended with sulphate; these cultures miner- archaeon in pure culture reported to degrade benzene coupled alized more than 90 % of the labelled benzene to CO2, al- to iron-reducing conditions (Holmes et al. 2011). though no sulphate depletion was confirmed (Edwards and Ferrogloblus placidus was able to degrade benzene, further Grbicgalic 1992). A later study using sediments from San accumulating benzoate but not phenol or toluene. Moreover, Diego Bay, California confirmed benzene oxidation coupled genes encoding the anaerobic degradation of benzoate were to sulphate reduction; in this study, benzene was metabolized upregulated during the growth of this pure culture on benzene within 55 days and new amendments increased its biodegra- versus growth on acetate and a putative carboxylase was dation (Lovley et al. 1995). These authors suggested a com- identified, suggesting a direct carboxylation of benzene to plete microbial oxidation to CO2 since no extracellular inter- form benzoate. mediates were found. Moreover, the addition of molybdate More recently, two Geobacter strains have been shown to (sulphate reduction inhibitor) completely stopped benzene degrade benzene in pure cultures coupled to the reduction of degradation (Lovley et al. 1995). iron. The Geobacter strain named Ben was isolated from a Further studies on marine and freshwater sediments sup- petroleum-contaminated aquifer in Minnesota and is most ported the oxidation of benzene under sulphate-reducing con- closely related to Geobacter daltonii (Zhang et al. 2012). ditions (Phelps et al. 1996; Weiner and Lovley 1998a, b; The strain also grew with Fe(III) oxide, Fe(III) nitrilotriacetic Anderson and Lovley 2000). Moreover, the inoculation of a acid, Fe(III) pyrophosphate and AQDS as electron acceptors. sulphate-reducing, benzene-oxidizing enrichment in sedi- The authors also reported that the genome of strain Ben ments from a petroleum-contaminated aquifer initiated Ann Microbiol (2015) 65:1201–1213 1207 successful anaerobic benzene degradation (Weiner and libraries during benzene degradation, as were sulphate- Lovley 1998a). Similarly, the addition of sulphate to some reducing Deltaproteobacteria and aceticlastic methanogens. Oklahoma sediments and to a gasoline-contaminated aquifer Based on these observations, a degradation pathway was in Bellingham, Washington significantly stimulated benzene proposed where the Cryptanaerobacter/Pelotomaculum phy- and BTEX degradation, respectively (Weiner et al. 1998; lotype assimilates most of the benzene, while hydrogen is Sublette et al. 2006). These observations were supported by formed and consumed by the Deltaproteobacteria and the an increase in the relative proportions of cyclopropyl fatty acetate produced is either converted to methane by aceticlastic acids, which in turn suggest an increase in the sulphate- methanogens or consumed by Epsilonproteobacterium reducing Desulfobacter species (Sublette et al. 2006). The (Herrmann et al. 2010;Vogtetal.2011) degradation of benzene in situ has also been successfully A later study identified phylotypes related to Pelobacter, demonstrated by the addition of sulphate (Anderson and Thermotogales and Methanolobus in an enrichment culture Lovley 2000; Cunningham et al. 2001). Moreover, the addi- prepared with a sample from an underground gas storage tion of sulphate to petroleum-contaminated aquifers has been aquifer with the ability to degrade BTEX (Berlendis et al. proposed as a more cost-effective and efficient bioremediation 2010). These authors also suggested syntrophy during the technique due to the much greater solubility of sulphate com- degradation of benzene. This was consistent with the results pared with oxygen (Weiner et al. 1998; Anderson and Lovley of a subsequent study on cultures derived from the Zeitz 2000;Lovley2001). aquifer where syntrophic interactions were also strongly sug- The first characterization of a sulphate-reducing marine gested since both acetate and hydrogen were found to be key consortium capable of anaerobic benzene degradation re- intermediates in benzene mineralization (Rakoczy et al. 2011). vealed a great diversity of microorganisms (Phelps et al. In this latter study on the Zeitz aquifer enrichment, the authors 1998). Phelps and colleagues characterized a consortium from used protein-SIP to analyse the carbon flux during the anaer- Guaymas, Mexico via clone libraries and found members of obic degradation of benzene. Mass spectrometric data allowed the Proteobacteria, Cytophagales and Gram-positive bacteria. quantification of the initial carbon source utilization and the Four clones (SB-9, 21, 29 and 30) were found to be related to metabolic intermediates. Members of Clostridiales, the Desulfobacteraceae, of which one was closely related to Deltaproteobacteria and Bacteroidetes/Chlorobi were identi- the aromatic degrader Desulfobacula toluolica strain Tol-2, fied as the functional groups of organisms in the quantitative while the other three were associated with the benzoate- analysis of carbon fluxes. These findings are consistent with degrader Desulfosarcina variabilis. Other clones were related previous results (Kleinsteuber et al. 2008). The Clostridiales to the sulphide oxidizer Thiomicrospira and to were confirmed to be involved in benzene degradation, puta- Campylobacter and Wolinella. Oka and colleagues (Oka tively fermenting benzene while fixing significant amounts of et al. 2008) later used DNA-SIP to show that clone SB-21 CO2.TheDeltaprotebacteria group used the metabolites re- incorporated most of the carbon from the 13C-labelled ben- leased during the anaerobic fermentation and a putative scav- zene into its DNA. This is constant with results obtained with enger group belonging to the Bacteroidetes/Chlorobi fed on clone BznS295, which was the dominant organism in another dead cells (Taubert et al. 2012). The phylogenetic classifica- sulphate-reducing, benzene-degrading marine culture and tion of the obtained proteins in the three groups revealed that found to be closely related to clone SB-21 (Musat and almost 95 % are related to the genera Desulfotomaculum and Widdel 2008), suggesting that these members of the Pelotomaculum within the Peptococcaceae family in the first Desulfobacteraceae are important players in the degradation group. The proteins in the Deltaproteobacteria group were of benzene coupled to sulphate reduction. found to be related to the Desulfobacca genus within the Other studies on cultures from a benzene-contaminated Synthrophaceae family, and the proteins in the Bacteroidetes/ aquifer near Zeitz, Germany identified members of the family Chlorobi group are related to the Rhodotermus genus within Peptococcaceae to be important players in benzene oxidation the Rhodothermaceae family. coupled to sulphate reduction via clone libraries (Kleinsteuber et al. 2008). This result was later confirmed by DNA-SIP (Herrmann et al. 2010) These organisms were found to be related to the Pelotomaculum and Cryptanaerobacter group Microorganisms identified in methanogenic conditions within Desulfotomaculum subcluster lh of the Peptococcaceae family within the Clostridia (Imachi et al. The process to produce methane involves a syntrophic asso- 2006). This phylotype along with an Epsilonproteobacterium ciation between proton-reducing bacteria and methanogenic incorporated carbon from 13C-labelled benzene in a DNA-SIP archaeons that consume hydrogen to obtain energy. experiment during the degradation of the contaminant Methanogens also keep the hydrogen concentration sufficient- (Herrmann et al. 2010). During benzene degradation, hydro- ly low for the reducing bacteria so they can still gain energy gen and acetate were detected as intermediates in clone from BTEX oxidation. 1208 Ann Microbiol (2015) 65:1201–1213

The degradation of BTEX coupled to methanogenesis has Microorganisms identified with other terminal electron been observed in several enrichment cultures derived from acceptors hydrocarbon-contaminated aquifers, sewage sludge and estu- arine muds (Kazumi et al. 1997; Weiner and Lovley 1998b; The degradation of benzene with the use of Mn(IV) as the Caldwell and Suflita 2000;DaSilvaandAlvarez2004). terminal electron acceptor was first documented a decade ago However, benzene degradation coupled to methanogenesis (Villatoro-Monzon et al. 2003) and subsequently confirmed can be a lengthy process (Kazumi et al. 1997) unless an by Villatoro-Monzón et al. (2008) in enrichments derived adapted consortium is present (Weiner and Lovley 1998b). from sediments of the Rhine river that had been shown in an Weiner and colleagues characterized a benzene-degrading earlier study by Cervantes et al. (2001)tobesuccessfulin methanogenic consortium composed of benzene- and terms of toluene degradation. BTEX and amorphous Mn(IV) phenol-degrading organisms, acetogenic organisms and oxide were amended to the enrichment as electron donor and aceticlastic methanogens (Weiner and Lovley 1998b). The acceptor, respectively, and benzene was the first of the BTEX latter belong to the Methanosaeta and Methanosarcina genera compounds to be degraded. Other studies have reported the in the Methanosarcinales division of the Archaea and are degradation of benzene using a graphite anode (Zhang et al. thought to perform the last steps in the degradation of hydro- 2010) and humic acids as electron acceptors (Cervantes et al. carbons since they have been constantly found in 2011). The use of the humic acid model compound AQDS as hydrocarbon-degrading consortia worldwide (Dojka et al. electron acceptor during the degradation of benzene was 1998; Ficker et al. 1999; von Wintzingerode et al. 1999; tested in two enrichments derived from hydrocarbon- Watanabe et al. 2002; Da Silva and Alvarez 2004; Kasai contaminated soil (PR) and sediment (ML) from two different et al. 2005; Struchtemeyer et al. 2005). The characterization locations in Mexico. The model compound enhanced the of hydrocarbon-degrading consortia has also revealed mem- degradation in both consortia, and the microorganisms ob- bers of the Desulfotomaculum within the Peptococcaceae, served in PR soil included two Gammaproteobacteria phylo- Synthrophus and aceticlastic methanogens (Dojka et al. types related to Pseudoxanthomonas and Pseudomonas,while 1998;Fickeretal.1999). It has been proposed that the members of the Betaproteobacteria, Gammaproteobacteria terminal steps of the hydrocarbon degradation are performed and Deltaproteobacteria, among others, were identified in by Syntrophus species since they produce hydrogen and the enrichment sediment ML (Cervantes et al. 2011). The acetate during the anaerobic oxidation of organic acids Geobacter strain Ben was also able to degrade benzene together with the aceticlastic methanogens. coupled to the reduction of AQDS (Zhang et al. 2012). AlaterstudybyUlrichandEdwards(2003) characterized a methanogenic benzene-degrading consortium that included members of the Desulfosporosinus (Peptococcaceae)andthe Anaerobic benzene degradation pathway Desulfobacterium (Desulfobacteraceae) genera, together with aceticlastic and hydrogenotrophic methanogens. These authors Three mechanisms have been proposed as the initial steps of suggested that bacteria related to the sulphate reducers initiate the anaerobic degradation pathway, namely, hydroxylation, the attack on benzene, while the Desulfosporosinus-related carboxylation and methylation (Coates et al. 2002)(Fig.2). bacteria would use the intermediates and produce acetate, hy- Recent studies on pure cultures suggest that the anaerobic drogen and carbon dioxide. Toluene and phenol were found as degradation pathway is dependent on the composition of the metabolites, suggesting that methylation was the initial step in microbial community. An initial hydroxylation step in the benzene degradation although a hydroxylation step was not Geobacter metallireducens is supported by the studies of discarded (Ulrich et al. 2005). Recent studies from Japan in- Zhang et al. (2012, 2013) on , carboxylation has been sug- volving DNA-SIP identified a deltaproteobacterium, Hasda-A, gested for Ferroglobus placidus and the enrichment dominat- as a key consortium member in benzene degradation coupled to ed by members of the Peptococcaceae from Zeitz, Germany methanogenesis in a culture derived from non-contaminated (Abu Laban et al. 2009, 2010;Holmesetal.2011)and soil (Sakai et al. 2009). The Hasda-A deltaproteobacterium methylation has been suggested for nitrate-reducing cultures was identical to the Desulfobacterium reported in the (Ulrich et al. 2005). The studies providing experimental sup- methanogenic consortium described earlier by Ulrich and port for each of the mechanisms are discussed in greater detail Edwards (2003), leading Sakai et al. (2009) to suggest in the following sections. syntrophic associations since hydrogenotrophic and aceticlastic methanogens were also detected in the enrichment. No metab- Hydroxylation olites were identified, and benzene was degraded completely to methane and carbon dioxide, which was supported by the The metabolites phenol, cyclohexanone and propionate concurrent mineralization of amended toluene benzoate and were detected under methanogenic conditions during the phenol (Masumoto et al. 2012). degradation of benzene in the mid-1980s, with water Ann Microbiol (2015) 65:1201–1213 1209

Fig. 2 Initial activation steps in the proposed anaerobic benzene degradation pathways that lead to benzoyl-CoA as the central metabolite. (A) Methylation, (B) hydroxylation, ©) carboxylation (modified from Vogt et al. 2011)

being suggested as the source of oxygen for the initial Methylation benzene oxidation (Vogel and Grbicgalic 1986; Grbicgalic and Vogel 1987). Later studies reported phenol and ben- The first evidence of benzene methylation was observed in zoate under methanogenic (Ulrich et al. 2005), nitrate-reducing cultures spiked with [13C]benzene (Ulrich sulphidogenic (Chaudhuri and Wiesmann 1995;Phelps et al. 2005). Labelled toluene and benzoate were recovered, et al. 2001) and iron-reducing conditions (Caldwell and leading the authors to suggest a pathway involving an initial Suflita 2000). Ulrich and Edwards (2005) suggested two methylation leading to toluene followed by a transformation different initial steps depending on the electron acceptor, to benzoate. This mechanism is supported by compound- i.e. hydroxylation under methanogenic conditions and specific isotope analyses (Zhang et al. 2002; Mancini et al. methylation under both nitrate-reducing and methanogen- 2003; Fischer et al. 2008; Mancini et al. 2008). ic conditions. Although phenol was reported as an inter- mediate for the Dechloromonas RCB strain (Chakraborty and Coates 2005), more recent studies strongly suggest Carboxylation that molecular oxygen is generated intracellularly from nitrate (Salinero et al. 2009;Weelinketal.2010). In A direct carboxylation of benzene leading to benzoate has also addition, phenol can also be produced abiotically if been suggested as an initial step in benzene mineralization; benzene-containing samples are in contact with atmo- benzoate has been observed as a metabolite in several studies spheric air (Kunapuli et al. 2008). of anaerobic benzene degradation (Caldwell and Suflita 2000; Nevertheless, Zhang and colleagues have suggested Phelps et al. 2001;Ulrichetal.2005). The recovery of labelled that phenol is an important metabolite during the growth benzoate from labelled benzene in freshwater and marine of an isolated Geobacter metallireducens strain on ben- sulphate-reducing cultures suggests benzene transformation zene under iron-reducing conditions (Zhang et al. 2013). (Caldwell and Suflita 2000;Phelpsetal.2001). Also, a This mechanism is supported by the upregulation of genes putative anaerobic benzene carboxylase was detected in pro- for phenol metabolism during benzene degradation and its tein extracts during the anaerobic degradation of benzene subsequent inhibition following the deletion of these under iron-reducing conditions (Abu Laban et al. 2010). Fur- genes, as mentioned above. In addition, these authors thermore, carboxylation has been suggested in the anaerobic confirmed that water is the source of oxygen for the benzene degradation by a pure culture of the hyperthermo- hydroxyl group. These results suggest that hydroxylation philic archaeon Ferroglobus placidus under iron-reducing may be the initial step for benzene degradation where conditions (Holmes et al. 2011). There was an increased Geobacter species are considered to be the key players expression of genes for anaerobic benzoate degradation dur- (Zhang et al. 2013). ing growth on benzene and a gene for a putative carboxylase 1210 Ann Microbiol (2015) 65:1201–1213 that is homologous to the one mentioned above was also Barbaro JR, Barker JF, Lemon LA, Mayfield CI (1992) identified (Holmes et al. 2011). Biotransformation of BTEX under anaerobic, denitrifying condi- tions—field and laboratory observations. J Contam Hydrol 11(3– 4):245–272 Barker JF, Patrick GC, Major D (1987) Natural Attenuation of Aromatic- Hydrocarbons in a Shallow Sand Aquifer. Ground Water Monit R 7(1):64–71 Future work Berlendis S, Lascourreges J-F, Schraauwers B, Sivadon P, Magot M (2010) Anaerobic biodegradation of BTEX by original bacterial The number and identification of consortia capable of anaer- communities from an underground Gas storage aquifer. 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