chlorinecontaining organics ( table  1 ) are often believed to originate exclusively from industrial pollution .
however , many living organisms ( e.g. marine sponges or terrestrial antagonistic microorganisms as a part of their defence mechanisms ) produce them naturally whereas chlorinated compounds are also released as a result of , for example , eruptions of volcanoes , forest fires and geothermal processes ( griebler et  al . , 2004 ; bengtson et  al . ,
nevertheless , it is their extensive industrial ( e.g. solvent , metal degreasing , rubber production ) and agricultural ( e.g. pesticide component ) application over the past 50 years that resulted in their deposition in various environments , especially in soils , groundwater aquifers and sediments ( bailey , 2001 ; meijer et  al . , 2003 ; barber et  al . , 2005 ;
due to their physicochemical properties ( table  1 ) , exposure to these compounds can have carcinogenic and lethal effects on biota .
therefore , the production and application of most of these compounds is no longer allowed in 90 countries since the stockholm convention in 2001 ( decision no . 2455/2001/ec , 2001 ; unep , 2005 ) .
remediation of soils and groundwater can be achieved via physicochemical methods such as thermal cleaning , chemical oxidation or adsorption of pollutants on activated carbon ( lai et  al . , 2007 ) , whereas there are no in situ remediation technologies for sediments other than complete removal of the contaminated sediment ( wenning et  al . , 2006 ) .
moreover , the high ecological disturbance that these physicochemical treatment methods can cause in the environment makes them unsustainable solutions in the long term ( wenning et  al . , 2006 ) .
other than harsh physicochemical treatments , a far more preferable option is bioremediation . during bioremediation chlorinated contaminants
phytoremediation , where plants are employed to assimilate , degrade , metabolize or detoxify chlorinated compounds , is an effective bioremediation method ( susarla et  al . , 2002 ) .
for example , poplar trees were shown to assimilate and degrade tce to 2,2,2trichloroethanol , trichloroacetic acid and dichloroacetic acid ( newman et  al . , 1997 ) .
recently , it has also been shown that the presence of these trees may stimulate the transformation of pce in the subsurface ( james et  al . , 2009 ) .
in this study , in the test location populated with hybrid poplar trees pce pollution was reduced by over 99% , in comparison with 2% removal in an unplanted control . moreover ,
pepo ( squash ) , were shown to extract milligrams of pcbs from soil in approximately 8 weeks time ( zeeb et  al . , 2006 ) . lately
the generation of transgenic plants to improve the phytoremediation of these pollutants resulted in several promising demonstrations of tce , 1,2dichloroethane ( dca ) and chlorophenol removal in several laboratory scale tests ( wang et  al .
, 2004 ; dowling and doty , 2009 ; james and strand , 2009 ) .
most fungi are robust organisms and are generally tolerant to high levels of pollution ( singh , 2006 ) .
fungal lignocellulolytic enzymes have been related to the degradation of various pollutants when used in combination with mediators and reactive radicals .
being the most commonly studied example , whiterot fungi are able to detoxify a wide range of pollutants including chlorinated organics , with lignin and manganese peroxidases ( tortella et  al .
sources , biological impacts and physicochemical properties of chlorinated organic compounds that have been reported to be degraded by 
hexachlorobenzene [ agency for toxic substances and disease registry ( atsdr ) , 2002 ] .
there are theoretically 209 different pcb congeners , although only about 130 of these were found in commercial pcb mixtures ( unep chemicals , 1999 ) .
chlorophenols [ agency for toxic substances and disease registry ( atsdr ) , 1999 ] . only pce is illustrated .
the bacterial degradation of chlorinated pollutants can be a result of fortuitous cometabolic conversion , or it may contribute to the energy metabolism of the degrading organism . during the latter metabolic processes ,
chlorinated compounds are used either as carbon source or as electron acceptor ( coupled to the oxidation of an electron donor ) , depending on the oxidation state of the compound .
although many chlorinated compounds may be transformed under aerobic conditions , the majority of polychlorinated compounds , such as those discussed in this review , are recalcitrant to aerobic degradation . due to the electronegative nature of the chlorine atom , oxidation of the carbon backbone in the chlorinated compound
becomes thermodynamically unfavourable ( wohlfarth and diekert , 1997 ) , especially in polychlorinated compounds . as a result
they serve as energetically favourable electron acceptors in microbial metabolism in anoxic environments such as sediments , subsurface soils and groundwater aquifers .
consequently , anaerobic bacteria , which can use these compounds as electron acceptors in a process termed organohalide respiration , are good candidates for bioremediation ( van eekert and schraa , 2001 ) . within the organohaliderespiring bacteria ,
dehalococcoides spp . and related isolates within the chloroflexi represent a special case in the anaerobic detoxification of halogenated organic contaminants .
it has been shown that several other bacteria belonging to the  and proteobacteria ( anaeromyxobacter , desulfuromonas , desulfomonile , desulfovibrio , geobacter , sulfurospirillum ) or to the lowgc grampositive bacteria ( desulfitobacterium , dehalobacter ) are also able to degrade chlorinated organic contaminants through organohalide respiration ( fig .  1 )
, none of these species are as specialized as dehalococcoides , and they are reported to grow as well , for example , by metal reduction , denitrification or fermentation .
alignment and phylogenetic analysis were performed with the arb software using the most recent release of the arbsilva project ( silva 96 ) ( ludwig et  al . , 2004 ;
pruesse et  al . , 2007 ) , and the tree was constructed using the neighbour joining method .
dehalococcoides is a genus of strictly anaerobic gramnegative bacteria that to the best of our knowledge are restricted to gaining energy from the reduction of chlorinated compounds by organohalide respiration .
isolates have an irregular , spherical shape ( approximately 0.5  m ) often referred to as coccoid .
electron acceptors such as oxygen , nitrate or sulfate has never been reported ( kube et  al . , 2005 ; seshadri et  al . , 2005 ) .
reductive dechlorination by dehalococcoides spp . occurs via the replacement of a chlorine atom in the chlorinated compound by hydrogen ( reductive hydrogenolysis ) and results in a net input of one proton and two electrons ( fig . 
2 ) ( holliger et  al . , 1998 ) . gibbs free energy ( g ) generated with reductive dechlorination of chlorinated compounds could range from 130 to 180  kj  mol per chlorine removed .
the redox potential thus generated is comparable to the redox potential of denitrification and higher than that generated by sulfate reduction . as a result
it was suggested that reductively dechlorinating bacteria could outcompete sulfate reducers and methanogens for reducing equivalents when the formation of reducing equivalents is rate limiting ( dolfing , 2003 ) .
are also capable of degrading chlorinated aliphatic compounds , i.e. 1,2dca , via socalled dihaloelimination . in this process
two neighbouring chlorine atoms are concurrently replaced via the formation of a double bond between the two carbon atoms .
dihaloelimination requires less h2 for the removal of chlorine atoms than reductive hydrogenolysis , thus its energy balance is more favourable under h2limited conditions ( smidt and de vos , 2004 ) .
reductive dechlorination of hexachlorobenzene ( hcb ) to pentachlorobenzene ( qcb ) . as the ecologists ' quest prevails to delve becking and beijerinck 's long running argument : 
everything is everywhere , but the environment selects ( beijerinck , 1913 ; becking , 1934 ) , the application of biomolecular tools , including the pcr amplification and sequencing of 16s ribosomal rna ( rrna ) genes from environmental samples , enables to study the full extent of microbial diversity and describe the biogeographical patterns exhibited by microorganisms at large spatial scales ( fierer and jackson , 2006 ; martiny et  al . , 2006 ) . with the growing interest in dehalococcoides ' presence and functioning in the environment ,
several studies were conducted using dehalococcoidesspecific 16s rrna genebased approaches in uncontaminated and chlorinated ethene contaminated sediments , soils and groundwater aquifers ( lffler et  al . , 2000 ; hendrickson et  al . , 2002 ; kittelmann and friedrich , 2008 ) .
currently , more than 100 16s rrna gene sequences of cultured and uncultured dehalococcoides spp . have been deposited to the database of the national center for biotechnology information ( ncbi ) .
1 ) ; however , various studies showed that this group is functionally very diverse ( maymogatell et  al .
, 1997 ; adrian et  al . , 2000 ; hendrickson et  al . , 2002 ; he et  al .
eight dehalococcoides strains have been isolated , mainly for their ability to degrade chlorinated ethenes ( table  2 ) .
functional differences in these isolates can be observed in the chlorinated compound transformed and in the transformation endproducts .
for example , the first isolate of the genusdehalococcoides ethenogenes strain 195 can completely dechlorinate pce to ethene , although degradation of vinyl chloride ( vc ) to ethene is cometabolic ( maymogatell et  al . , 1997 ) .
dehalococcoides ethenogenes strain 195 can also dechlorinate hcb to 1,3dcb ( dichlorobenzene ) , 1,4dcb , 1,2dcb and 1,3,5tcb ( trichlorobenzene ) .
cbdb1 dechlorinates hcb to 1,3dcb , 1,4dcb and 1,3,5tcb , and recently also transformation of pce and tce to transdce was observed ( adrian et  al . , 2007b ) .
are difficult to maintain in pure culture ( maymogatell et  al . , 1997 ; adrian et  al . , 2000
; he et  al . , 2003 ) ; they are more easily maintained in a microbial community , on which they depend for h2 supply , as long as ideal growth conditions are provided ( duhamel et  al .
examples include chlorinated ethene transforming cornell ( maymogatell et  al . , 1997 ) , victoria ( hendrickson et  al . , 2002 ) , pinellas ( harkness et  al . , 1999 ) , kb1 ( duhamel et  al . , 2002 ) and anas cultures ( richardson et  al . , 2002 ) .
, two other distantly related isolates within the chloroflexi have recently been obtained ( fig .  2 ) .
dehalobium chlorocoercia df1 is able to dechlorinate a variety of pcbs ( may et  al .
this microorganism dechlorinates polychlorinated alkanes ( yan et  al . , 2009 ) . like dehalococcoides spp . , both isolates are strictly hydrogenotrophic
several enrichment studies showed the presence of dehalococcoides spp . in different locations and environments in the northern hemisphere (
dehalococcoidescontaining enrichment cultures originating from river sediments have been shown to dechlorinate pcb and dioxin congeners , pce , tce and a number of chlorinated benzenes ( ballerstedt et  al . , 2004 ; yoshida et  al .
, 2005 ; bedard et  al . , 2007 ; bunge et  al . , 2007 ;
besides sediment enrichments , dechlorination by dehalococcoides was also reported in groundwater aquifers ( bowman et  al .
, 2006 ; brgmann et  al . , 2008 ; imfeld et  al . , 2008 ; lee et  al . , 2008 ; himmelheber et  al . ,
2009 ) and a denitrifying membranebiofilm reactor ( chung et  al . , 2008 ) .
few studies have demonstrated that the bioaugmentation with reductively dehalogenating cultures can result in complete dechlorination of pce and tce to ethene ( ellis et  al .
, 2000 ; major et  al . , 2002 ; lendvay et  al . , 2003 ; scheutz et  al . ,
in pure and enrichment cultures are in the range of 0.20.4  day under laboratory conditions ( maymogatell et  al . , 1997 ; cupples et  al . , 2003 ;
additionally , quantitative analyses of the dehalococcoides spp . 16s rrna gene at chlorinated ethene bioremediation sites ( soil and groundwater ) revealed abundances of 1010 copies per gram material ( lendvay et  al .
recently , a groundwater bioremediation simulation study showed that growth rates obtained in laboratory conditions could also be replicated in largescale experiments , which resulted in up to 10 16s rrna gene copies l ( vainberg et  al . , 2009 ) .
hence , these pilot as well as fieldscale bioremediation tests with dehalococcoidescontaining cultures offer promising results for the further use of these microorganisms . in spite of all the information obtained in physiological studies
very little is known about the diversity , distribution and functioning of dehalococcoides in different environments although they were detected at several contaminated locations .
hendrickson and coauthors have demonstrated the presence of dehalococcoides spp . in soil and groundwater samples from 24 sites scattered throughout north america and europe ( hendrickson et  al . , 2002 ) .
up to 200  m pce could be dechlorinated , and complete dechlorination to ethene could be correlated to the presence of dehalococcoides spp . in the sampling locations .
recently , we conducted a largescale survey focusing on presence , activity and dechlorination potential of dehalococcoides spp . in river sediments and floodplain soils from different polluted locations in europe ( fig . 
almost all of the tested sediment and soil samples showed the capacity to dechlorinate hcb and/or chlorinated ethenes irrespective of the in situ contaminant levels .
nevertheless , the hcb transformation rates observed in the laboratoryscale microcosms and the number of 16s rrna gene copies of dehalococcoides spp . in the environmental samples
relative abundance was furthermore shown to change significantly along temporal and spatial gradients , but was also found to be influenced by other environmental factors such as water temperature ( taet  al . , 2009 ) .
summary of results from the locations studied byta ( 2009 ) with cultivationdependent and independent molecular methods .
detection with 16s rrna and/or 16s rrna genetargeted methods ; : hcb transformation ; : chlorinated ethene transformation ; (  ) no detection or no transformation ; ( + /++/+++ ) low to high rrna copies or long to short lag phases in hcb and chlorinated ethene transformation ; na : not available ; ( ) soil and ( ) river sediment sample from schnberg , germany .
map was redrawn from openstreetmap ( http://www.openstreetmap.org ) . as nonfermentative microorganisms dehalococcoides spp . and
their organohaliderespiring relatives dehalobium chlorocoercia df1 and dehalogenimonas lykanthroporepellens dc9 depend on the h2 supply from other microorganisms for their growth ( smidt and de vos , 2004 ; may et  al .
recently , it has also been suggested that the activity of dehalococcoides spp . in in situ conditions
is linked to the performance of fermentative communities ( rling et  al . , 2007 ) .
therefore , it is crucial to have insight in factors affecting nutrient fluxes and microbial communities involved in carbon , nitrogen and sulfur ( c ,
n , s ) cycling in the river basins to be able to understand the survival and functioning of dehalococcoides spp . in different geographical locations . because there are considerable differences between dechlorination capabilities of the known dehalococcoides strains despite 16s rrna identities of >  99% , their sole presence based on the detection of the 16s rrna gene in an environment does not guarantee successful in situ dechlorination of a specific pollutant .
consequently , molecular tools that target metabolic activities of the entire microbial communities in the environment are needed to have a canonical assessment of the conditions .
our knowledge gap concerning the properties of dehalococcoides spp . is closing rapidly with the developments in highthroughput sequencing technologies .
nc_002936 ) and strain cbdb1 ( nc_007356 ) genomes are approximately 1.47 and 1.39 million base pairs ( mbp ) respectively .
both genomes comprise single circular chromosomes with 1591 predicted protein coding sequences ( cds ) in strain 195 ( seshadri et  al . , 2005 ) and 1458 cds in strain cbdb1 ( table  3 ) . up
to 1217 of the cds from strain cbdb1 have orthologous genes in d. ethenogenes strain 195 ( 83.5% ) ( kube et  al . , 2005 ) .
strain bav1 ( nc_009455 ) has a genome of 1.34  mbp with 1385 cds based on information provided in the integrated microbial genomes ( img ) database , release march 2009 ( markowitz et  al . , 2008 ) .
for example , one copy of each rrna gene is present in all dehalococcoides genomes ( kube et  al . , 2005 ; seshadri et  al . ,
comparative analysis of available dehalococcoides genomes showed that 70% of all genes in these genomes have a high sequence and contextual conservation ( mcmurdie et  al . , 2008 ) .
interestingly , d. ethenogenes strain 195 possesses a nitrogenaseencoding operon , which is missing in strain cbdb1 . even though this finding suggests that d. ethenogenes strain 195
can fix nitrogen , diazotropic growth of the dehalococcoides strains has not yet been reported .
comparison of wholegenome sequence statistics for reductively dechlorinating bacteria as presented in integrated microbial genomes ( img / m ) database , march 2009 ( markowitz et  al . , 2008 ) .
genes : total gene count ; cds : coding sequences ; rna : number of rrna , trna and other rna genes ; 16s : number of 16s rrna gene copies ; orthologues : number of genes in orthologues ; paralogues : number of genes in paralogues ; rdh genes : confirmed and predicted reductive dehalogenaseencoding genes .
different dehalococcoides strains contain different numbers of rdh genes that encode protein , which have been proven or predicted to catalyse the dechlorination reaction . when compared with the genomes of other dechlorinating bacteria , dehalococcoides have the highest number of rdh genes in their genomes ( table  3 ) .
genomes of strains 195 , cbdb1 and bav1 have 17 , 32 and 10 rdh genes , respectively , whereas only seven rdh genes were identified in the genome of desulfitobacterium hafniense dcb2 , four rdh genes in d. hafniense y51 and two rdh genes in geobacter lovleyi sz and anaeromyxobacter dehalogenans ( thomas et  al . ,
the draft genome of strain vs contains the highest number of rdh genes ( 36 fulllength genes ) ever found in a single bacterial genome ( mcmurdie et  al . , 2008 ) .
similarly , 14 and 19 rdh genes were detected via pcr amplification in dehalococcoides sp .
strains fl2 and dcmb5 respectively ( holscher et  al . , 2004 ; bunge et  al . ,
twelve rdh genes from strain cbdb1 have orthologues in d. ethenogenes strain 195 genome with 86.495.4% sequence identity . in d. ethenogenes strain 195 and strain cbdb1 genomes almost all of the rdh genes ( except det0079 , tce reductive dehalogenase tcea in d. ethenogenes strain 195 and cbdba1583 in strain cbdb1 ) were found to be located in close proximity to genes for transcription regulators , and were predicted to be transcribed in the direction of dna synthesis , which suggests tight regulation of rdh activity ( kube et  al . , 2005 ; seshadri et  al . ,
only two rdh genes from strain 195 , det0079 and det0318 , have been characterized as tce ( tcea ) and pce ( pcea ) reductive dehalogenases respectively ( fung et  al . , 2007 ) .
the cbdba84 gene from strain cbdb1 was recently designated as a chlorobenzene reductive dehalogenase ( cbra ) , which is involved in dechlorination of 1,2,3,4tecb and 1,2,3tcb ( adrian et  al .
additionally , two vc reductase genes were identified from strain bav1 ( bvcra , dehabav1_0847 ) ( krajmalnikbrown et  al . , 2004 ) and strain vs ( vcra , genbank accession no .
can not be inferred from dehalococcoides phylogeny , detection methods based on processspecific biomarkers are necessary to describe the bioremediation capacity and activity of dehalococcoides in the environment .
therefore , genes like rdhs and the corresponding gene products that are specific to functions of interest can serve as useful biomarkers in monitoring of different dehalococcoides activities . in the past years microarrays were shown to be useful tools for such monitoring activities and characterization of microbial communities ( zhou , 2003 ; wang et  al . , 2009 ) .
furthermore , functional gene arrays ( fgas ) , which target functional genes such as nitrogenases , cellulases etc .
, allow fast and comprehensive analysis of metabolic potential and activity of microbial communities in the environment by targeting a large number of genes or their transcripts in one single experiment ( wu et  al .
, 2001 ; taroncheroldenburg et  al . , 2003 ; steward et  al . , 2004 ; zhou et  al . , 2008 )
up to date the most extensive fga platform is the geochip ( he et  al . , 2007 ) , which targets approximately 10  000 catabolic genes involved in major biogeochemical cycles , including those of carbon , nitrogen and sulfur , as well as organic pollutant degradation .
analysis of hcbcontaminated sediments in the ebro river basin ( spain ) using the geochip amended with probes targeting 153 rdh genes showed that rdh gene diversity changed significantly between different sampling locations ( taet  al . , 2009 ) .
more specifically , sediment samples taken at a site with high hcb pollution ( lacorte et  al . , 2006 ) were dominated by rdh genes of dehalococcoides spp .
in contrast samples , which were characterized by more diffuse pollution with a broader range of contaminants , a wide spectrum of rdh genes was detected including those from various other organohaliderespiring microorganisms ( fig . 
however , it should be noted that microarrays can only detect known sequences , which can cause an underestimation of functional gene diversity and abundance in environments for which limited sequence information is available .
application of fgas in combination with newly developed techniques such as highthroughput nongelbased proteomics ( maron et  al . , 2007 ) and
strain cbdb1 transcriptomes suggested continuous transcription of rdh genes such as tcea ( johnson et  al . , 2008 ) and cbra ( wagner et  al .
, 2009 ) during different growth phases . as a result gene transcripts of such genes can be studied using transcriptomic techniques with fgas , in combination with proteomics methods ( morris et  al . , 2006 ;
morris et  al . , 2007 ) to identify the proteins with significant functional impact .
hierarchical cluster analysis of rdh gene profiles based on geochip functional gene array hybridization signals for samples from flix and rice fields ( rf , river delta ) in the ebro river .
hcb is reported to be the dominant chlorinated contaminant in ebro 's basin where location flix bares the highest hcb pollution ( lacorte et  al . , 2006 ) .
the greyscale intensity indicates differences in hybridization signal intensity , with black representing the strongest signals .
samples are represented according to sampling month , year and sampling depth ( i.e. f06d5 : february 2006 depth 05  cm ; j04d15 : june 2004 depth 1015  cm )
. for accession numbers of rdh gene targets , see ta and colleagues ( 2009 ) .
the broad aim of systems microbiology is to define and understand the relationships between the individual components that build a cellular organism , a community and an ecological niche ( vieites et  al . , 2009 ) . as a result , in the past , the focus of systems microbiology was on microbial isolates or enrichments ( mchardy and rigoutsos , 2007 ) . to date , the majority of the research conducted in the field of reductive dechlorination has been predominantly focused on the identification of genes and proteins directly responsible for the dechlorination process ( cupples et  al . , 2003 ; muller et  al . , 2004 ; holmes et  al .
, 2006 ; adrian et  al . , 2007a ; mcmurdie et  al . , 2007 ; west et  al . , 2008 ; wagner et  al . ,
these experimental studies , so far , allowed the analysis and characterization of several key genes .
however , it is becoming evident that to understand microbial functions or functioning of microbial communities one must study the entire system ( vieites et  al . , 2009 ) .
the body of research summarized in this review also supports this idea and suggests that with biomolecular assays targeting ribosomal and processspecific functional genes such as those encoding reductive dehalogenases , it will remain difficult to understand the full extent of the process , since the dechlorination process comprises an integral part of a complex web of metabolic and regulatory interactions ( rahm et  al . , 2006 ; west et  al .
the application of novel , more comprehensive methods like whole genome shotgun ( wgs ) sequencing of environmental dna and mrna ( functional metagenomics ) ( tringe et  al . , 2005 ; kalyuzhnaya et  al . ,
2008 ) , the establishment of largescale databases which contain metagenomic data from different environments ( seshadri et  al . , 2007 ;
, 2009 ; vogel et  al . , 2009 ) as well as the development of new computational resources for comparative ( meta)genomic analyses ( peterson et  al .
, 2001 ; alm et  al . , 2005 ; markowitz et  al . ,
2006 ) enable us to develop and analyse data sets ( and microbial networks ) which so far are believed to be the closest to the actual environmental situations .
thus , today , it can be proposed to leave reductionist approaches that are limited to only one or a few selected biomarkers , and to study reductive dechlorination and the function of dehalococcoides spp . in larger communities and in the environments in which they belong .
as the functional properties of such communities are elucidated , we will be able to assess the true role and importance of dehalococcoides spp .
 summarythe fate and persistence of chlorinated organics in the environment have been a concern for the past 50 years . 
 industrialization and extensive agricultural activities have led to the accumulation of these pollutants in the environment , while their adverse impact on various ecosystems and human health also became evident . 
 this review provides an update on the current knowledge of specialized anaerobic bacteria , namely  
 dehalococcoides spp . , which are dedicated to the transformation of various chlorinated organic compounds via reductive dechlorination . 
 advances in microbiology and molecular techniques shed light into the diversity and functioning of dehalococcoides spp . in several different locations . 
 recent genome sequencing projects revealed a large number of genes that are potentially involved in reductive dechlorination . 
 molecular approaches towards analysis of diversity and expression especially of reductive dehalogenaseencoding genes are providing a growing body of knowledge on biodegradative pathways active in defined pure and mixed cultures as well as directly in the environment . 
 moreover , several successful field cases of bioremediation strengthen the notion of dedicated degraders such as dehalococcoides spp . as key players in the restoration of contaminated environments 
 . 
Pollution of chlorinated compounds and their bioremediation
The little bacteria that can: the genus 
Discoveries from 
Future perspectives: reductive dechlorination, systems microbiology and microbial networks