manganeseoxidizing bacteria oxidize mn to form insoluble mn and/or mn oxides , and this far best studied bacterial species have been isolated from manganesecontaining environments such as mnoxide encrusted pipelines , manganesecontaining water pipes and aquifers , as well as from marine sediments ( schweisfurth , 1973 ; nealson and ford , 1980 ; emerson and ghiorse , 1992 ) .
however , the natural niche of manganeseoxidizing bacteria appears not only to require the presence of manganese , but often also features a marked availability of usually recalcitrant organics in nature .
thus , an essential role of these bacteria in oxidation of organic material in organicrich environments has been postulated ( sunda and kieber , 1994 ) . moreover ,
mnoxidizing bacteria have been detected in environmental samples contaminated with recalcitrant xenobiotics , such as polycyclic aromatic hydrocarbons or methyl tertbutyl ether ( bruns et  al .
, 2001 ; bodour et  al . , 2003 ; raynal and pruden , 2008 ) . to test the potential use of bacterial manganese oxidation to drive the degradation of recalcitrant polycyclic aromatic organic compounds , 17ethinylestradiol ( ee2 ) was chosen as target compound .
ee2 is a persistent synthetic estrogen and is often present as a trace pollutant in waste and drinking water bearing the proven risk of undesirable hormonal effects on animals and humans ( thomas et  al . , 2001 ) .
the persistence of ee2 has prompted a number of attempts to achieve degradation of ee2 by bacteria , e.g. by use of rhodococcus zopfii , rhodococcus equi and sphingobacterium sp .
all these bacteria require ee2 to be present at milligram levels . in situ , however , only trace concentrations of the pollutants are normally present in waste and drinking waters , which are unlikely to sustain bacterial growth , thus limiting the use of these bacteria for degradation of e.g. trace amounts of ee2 .
it has recently been demonstrated that commercially available manganese oxides as powder or as specifically produced nanoparticles can mediate removal of ee2 ( de rudder et  al . ,
the main consideration for using biogenic mn oxides produced by mnoxidizing bacteria , instead of chemical mn oxides as used before by de rudder and colleagues ( 2004 ) , was to explore the possibility of working at significantly lower ( micromolar ) concentrations of mn .
it was shown before that biogenic mn oxides have different structure and surface areas compared with chemical oxides ( saratovsky et  al . , 2006 ) , and this might bring about their increased activity towards ee2 .
second , we are aiming at construction of a selfregenerating system allowing cyclic oxidation and reduction of mn oxides linked to continuous concomitant degradation of ee2 . in this study
, we propose an efficient strategy for the removal of trace levels of xenoestrogens from environmental samples by biogenic generation of highly oxidized mn oxides by manganeseoxidizing bacteria , which in turn drive the indirect oxidation and cleavage of ee2 . as degradation of substituted phenols , fluoroquinolone antibiotics , aniline and other aromatic amines by manganese oxides
have also been reported ( stone , 1987 ; laha and luthy , 1990 ; zhang and huang , 2005 ) , the use of highly oxidized biogenic mn oxides can be expanded to the degradation of other organic pollutants .
the use of manganeseoxidizing bacteria can be a starting point for the development of a more general strategy for the purification of waste and drinking water from estrogens and other tracelevel organic contaminants .
in order to test if manganese oxides derived from oxidation of mn by manganeseoxidizing bacteria may mediate degradation of ee2 , four different microbial strains namely leptothrix discophora , pseudomonas putida strains lmg 2321 , 2322 and 2323 , which are known to catalyse oxidation of mn , were tested for ee2 degradation .
these strains were grown on a complete medium with glucose as a carbon source and with a small amount of ee2 ( 0.5  m ) in presence or absence of different concentrations of mn ( 25 , 50 , 75 , 100  m ) .
all tested strains have shown the ability to oxidize mn , as observed by the decrease of mn content ( table  1 ) and also by accumulation of mn oxides , visible as insoluble brown or orange particulate matter .
the amount of ee2 degraded by l.  discophora and lmg 2322 strains was proportional with the oxidized mn . without manganese only a slight decrease of ee2 ( around 10% ) was detected ( table  1 ) , which was independent from the culture growth and may be attributed to adsorption of ee2 to biomass .
all the remaining levels of ee2 were found to be very stable in the mnfree bacterial cultures even after 4 weeks of incubation ( data not shown ) .
elevated concentrations of mn ( higher than 100  m ) negatively affected oxidation of mn and the degradation of ee2 ( data not shown ) , which again points at mn oxidation to be a prerequisite to the degradation of ee2 .
ee2 degradation was measured by hplc analysis , and adsorption of ee2 to biogenic manganese oxides were determined as described in experimental procedures section .
the initial concentrations of mn used in this study are known from the earlier studies to induce mn oxidation , but were by far represented in excess compared with 0.5  m ee2 . in order to establish a lower limit of mn which starts to catalyse degradation of ee2 , lmg 2322
was grown with 0.5  m of ee2 and 1 , 10 and 50  m mn .
degradation of ee2 is dependent on the amount of mn present in the medium and that multifold excess of mn was required ( fig .  1 ) .
most of ee2 was degraded in the late stationary phase and thus was not linked to the growth of the bacteria ( fig .  1 ) .
moreover , the degradation of ee2 occurred in time after mn oxidation : for the culture with 50  m of mn most of ee2 was degraded in the period between 24 and 36  h , whereas most of mn was oxidized earlier , in the period between 18 and 24  h ( fig .  1 ) .
this delay is likely to be linked to the primary accumulation of significant amounts of biogenic mn oxides , which then start to react with ee2 .
thus , degradation of ee2 is a slow chemical process that is not dependent on the presence of viable bacterial cells , but rather on the amount of biogenic mn oxides produced .
influence of microbial activity on mndependent degradation of ee2 was tested by addition of sodium azide , a known inhibitor of electron transportlinked oxidation .
pseudomonas putida was grown in presence of 100  m of mncl2 until orange coloured mn oxides were visible and ee2 and sodium azide at a concentration of 1  mm were added .
addition of sodium azide still allowed 80% degradation of ee2 , whereas without azide complete degradation of ee2 occured .
we suspect that the 20% inhibition caused by azide could be linked to inability of the azidetreated cultures to regenerate mn oxides .
a. growth of p.  putida in the presence of 0.5  m of ee2 and 1 , 10 and 50  m of mn . 
b. dynamics of mn oxidation .  c. dynamics of degradation of 0.5  m of ee2 with different initial concentrations of mn . for each measurement , data represent a mean value obtained from three identical cultures . earlier it was suggested that ee2 may be adsorbed in significant amounts to chemically synthesized manganese oxides ( de rudder et  al . ,
2004 ) . in order to rule out the adsorption of ee2 to insoluble mn oxides generated by the mnoxidizing bacteria and thus to distinguish it from oxidation of ee2 ,
cultures were treated with ascorbic acid , which is known to reduce higher oxidation states of mn back to readily soluble mn ions ( stone and morgan , 1984 ) , thereby releasing potentially adsorbed ee2 . with few exceptions ( e.g. lmg 2321 at 25  mm mn ,
lmg 2323 at 50  m and 100  m mn ) , where small amounts of apparently adsorbed ee2 were recovered after ascorbic acid treatment , in all other mnsupplied samples no additional ee2 was released after treatment with ascorbic acid ( table  1 ) .
ascorbic acid by itself did not affect the concentration of ee2 , as shown by ee2 concentrations of mnfree control cultures before and after the treatment with ascorbic acid ( data not shown ) .
moreover , upon careful examination of hplc spectra an intermediary product of ee2 was detected at low levels and with a retention time of 9.8 . because this product disappeared in a manganesedependent fashion and had no measurable estrogenic effect , its structure was not further examined . therefore , decreased levels of ee2 in the respective mncontaining cultures are accomplished by true degradation , rather than by adsorption to the insoluble mn oxides that are formed . as oxidation of ee2 by mn oxides may result in production of metabolites with undesirable estrogenic activity , we performed yeast estrogen screen ( yes ) bioassays .
the yes assay is based on the use of a recombinant yeast strain that harbours a human estrogen receptor gene and is used to measure the estrogenic activity of environmental samples ( pierrat et  al . , 1992 ;
estrogenic response of the culture of the strain lmg 2322 grown with 0.5  m ee2 and 100  m of mn was comparable with the one of negative control , indicating that no residual estrogenic activity in this mncontaining sample was present where ee2 was found to be eliminated ( fig .  2 ) .
by contrast , in the corresponding culture grown in the absence of mn where ee2 was still present , strong estrogenic response was found as expected ( fig .  2 ) .
estrogenic responses of cultures of p.  putida 2322 grown with 0.5  m ee2 and with / or without 100  m of mncl2 ( black or white squares , correspondingly ) ( b ) .
estrogenic responses of distilled water ( black squares ) and a solution of 0.5  m ee2 ( white squares ) were used as corresponding controls ( a ) .
it was shown that degradation of ee2 may be linked to the extracellular enzymatic activity in other organisms , such as to the manganese peroxidase and multicopper oxidase activities of white rot fungi ( suzuki et  al . , 2003 ) .
this is unlikely to be the case for the manganeseoxidizing bacteria tested here for the following reasons : ( i ) the rate of ee2 degradation is strongly correlated with the amount of mn oxidized and such correlation is normally unlikely to be linked to the enzymatic activity ; ( ii ) oxidation of ee2 does not coincide in time with the oxidation of mn , when the mnoxidizing enzyme is produced , but it is delayed and rather linked with the period when most of mn has been oxidized and the bulk of mn oxides have been accumulated ; ( iii ) enzymatic treatment of the mn and ee2containing culture with pronase at two different concentrations of the enzyme ( 100  m , 1  mm ) with the aim to abolish the extracellular enzymatic activity has not resulted in any noticeable inhibition of the ee2 degradation ( data not shown ) . to conclude , manganeseoxidizing bacteria are of particular biotechnological interest , not only with respect to their use in the wellestablished biological treatment of manganesecontaining waters , but also for bioremediation of environments contaminated with trace concentrations of recalcitrant organic pollutants .
we presented the indirect oxidation of ee2 by biogenic manganese oxides as a concept of a novel biological process aiming to eliminate trace concentrations of organic pollutants from environmental samples .
the initial attack of highly oxidized manganese oxides on ee2 is likely to address chemical features of the phenolic groups also found in other polycyclic aromatic compounds , which could thus likewise be degraded by highly oxidized manganese .
the breakdown of other compounds like amines , aromatic amines and xenobiotics with sulphydryl groups were also reported to be chemically degraded by mn oxides , and are thus also likely to be degraded in the presence of manganeseoxidizing bacteria and mn .
our future studies will be focused on designing a bioprocess aiming at the degradation of ee2 by mnoxidizing bacteria . a serious step to consider
is that the lowest concentration which still allows efficient degradation of ee2 found in this study was 50  m , which is still five times higher than the allowed concentrations of manganese in drinking water .
however , designing a bioreactor with alternating conditions would result in both complete degradation of ee2 and complete oxidation of mn .
the oxidized insoluble manganese oxides would then precipitate and could be this way removed from the water .
generally , these results draw attention to the potential value of microbial extracellular oxidative processes ( like bacterial manganese oxidation ) for biotechnological processes aiming at degradation of trace pollutants .
leptothrix discophora ( lmg 8142 , belgian coordinated collections of microorganisms ) and p.  putida strains mnb1 ( lmg 2321 ) , mnb6 ( lmg 2322 ) and mnb29 ( lmg 2323 ) were cultivated on a medium described by boogerd and de vrind ( 1987 ) . to assess degradation of ee2 and
to determine manganese oxidation by the manganeseoxidizing bacteria , two types of overnight precultures were set without and with filtersterilized manganese chloride at a concentration of 25  m . on the following day ,
50  ml of the medium with ee2 ( 0.5  m ) and mncl2 at different concentrations ( 0 , 25 , 50 , 75 , 100  m ) were inoculated , bacterial growth ( by measuring od600 ) was monitored and concentrations of ee2 and mn were measured .
mn oxidation was monitored in 2  ml aliquots from the cultures , which were centrifuged at 13  400  g for 30  min .
after mn concentrations were determined by using the formaldoxime assay described by bartley and colleagues ( 1957 ) .
briefly , 3  ml of the supernatant was mixed with 1  ml of formaldoxime hydrochloride solution ( sigmaaldrich , 0.5  g dissolved in 10  ml of distilled water ) .
culture aliquots were filtered through a 0.22  m filter prior to hplc analysis . in order to test for potential adsorption of ee2 to particulate mn oxides generated by bacterial oxidation of mn , ascorbic acid ( 0.11  m ) was added to 5  ml culture aliquots , and samples were shaken for 15  min at 30c .
the samples were analysed for ee2 before and after the treatment with ascorbic acid , which , being a strong reductant , was expected to release adsorbed ee2 by regenerating soluble mn from insoluble mn oxides . ee2 and its potential metabolites were analysed by reversed phase hplc with an asi100 autosampler , p580 pump and sth585 column oven ( dionex , sunnyvale , ca , usa ) on an agilent zorbax sbc18 column ( 150  mm    4.6  mm , 5  m ) .
elution was performed at 25c and a flow rate of 1  ml / min with solvent a ( 100% acetonitrile ) and solvent b ( milliq  +  0.1% formic acid ) .
the injection volume was 50  l and the elution program was as follows : 2  min isocratic at 85% b , linear gradient to 60% b in 5  min , linear to 35% b in 15  min , linear to 0% b ( or 100% a ) in 3  min follow by 5  min isocratic 100% a , linear gradient to 85% b and isocratic at 85% for 5  min .
detection of ee2 , e2 and e3 was performed at an excitationemission wavelength of 280310  nm ( yoon et  al . , 2003 ) by rf2000 fluorescence detector ( dionex ) and
standards ranging from 2 to 1000  g  l for ee2 for calibration were prepared , starting from individual ethanol stock solution containing 1  g  l of ee2 .
the yes assay is based on the use of a recombinant yeast strain that harbours a human estrogen receptor gene ( pierrat et  al . , 1992 ) and is used to measure the estrogenic activity of environmental samples ( baker , 2001 ) .
the bacteria were grown either with manganese and ee2 or with manganese alone ( control conditions ) for 72  h ( until latestationary stage of growth ) as described above .
one millilitre of each culture was then filtered through a 0.22  mfilter and filtrates were used for the yes assay .
the yes assay was performed as described ( de boever et  al . , 2001 ) in 96well plates in which 10  l of filtrates diluted twice with dimethyl sulfoxide were incubated with 240  l of the genetically modified saccharomyces cerevisiae at an optical density of 0.25 at 610  nm .
additional plates with serial dilutions of either the medium alone ( negative control ) , or of medium supplemented with ee2 in dmso ( positive control ) were made and equally tested .
estrogenic activity responses were recorded as the absorbance at 540  nm divided by the optical density at 630  nm [ ( a540/a630)net ] ( sunrise , plate reader , tecan , grdig , austria ) . based on the data obtained ,
the data were fitted by a fourparametric logistic model using the marquardtlevenberg algorithm ( sigmaplot 4.0 ; spss inc . ,
chicago , il , usa ; de boever et  al . , 2001 ) .
leptothrix discophora ( lmg 8142 , belgian coordinated collections of microorganisms ) and p.  putida strains mnb1 ( lmg 2321 ) , mnb6 ( lmg 2322 ) and mnb29 ( lmg 2323 ) were cultivated on a medium described by boogerd and de vrind ( 1987 ) . to assess degradation of ee2 and
to determine manganese oxidation by the manganeseoxidizing bacteria , two types of overnight precultures were set without and with filtersterilized manganese chloride at a concentration of 25  m . on the following day ,
50  ml of the medium with ee2 ( 0.5  m ) and mncl2 at different concentrations ( 0 , 25 , 50 , 75 , 100  m ) were inoculated , bacterial growth ( by measuring od600 ) was monitored and concentrations of ee2 and mn were measured .
mn oxidation was monitored in 2  ml aliquots from the cultures , which were centrifuged at 13  400  g for 30  min .
after mn concentrations were determined by using the formaldoxime assay described by bartley and colleagues ( 1957 ) .
briefly , 3  ml of the supernatant was mixed with 1  ml of formaldoxime hydrochloride solution ( sigmaaldrich , 0.5  g dissolved in 10  ml of distilled water ) .
culture aliquots were filtered through a 0.22  m filter prior to hplc analysis . in order to test for potential adsorption of ee2 to particulate mn oxides generated by bacterial oxidation of mn , ascorbic acid ( 0.11  m ) was added to 5  ml culture aliquots , and samples were shaken for 15  min at 30c .
the samples were analysed for ee2 before and after the treatment with ascorbic acid , which , being a strong reductant , was expected to release adsorbed ee2 by regenerating soluble mn from insoluble mn oxides . ee2 and its potential metabolites were analysed by reversed phase hplc with an asi100 autosampler , p580 pump and sth585 column oven ( dionex , sunnyvale , ca , usa ) on an agilent zorbax sbc18 column ( 150  mm    4.6  mm , 5  m ) .
elution was performed at 25c and a flow rate of 1  ml / min with solvent a ( 100% acetonitrile ) and solvent b ( milliq  +  0.1% formic acid ) .
the injection volume was 50  l and the elution program was as follows : 2  min isocratic at 85% b , linear gradient to 60% b in 5  min , linear to 35% b in 15  min , linear to 0% b ( or 100% a ) in 3  min follow by 5  min isocratic 100% a , linear gradient to 85% b and isocratic at 85% for 5  min .
detection of ee2 , e2 and e3 was performed at an excitationemission wavelength of 280310  nm ( yoon et  al . , 2003 ) by rf2000 fluorescence detector ( dionex ) and
standards ranging from 2 to 1000  g  l for ee2 for calibration were prepared , starting from individual ethanol stock solution containing 1  g  l of ee2 .
the yes assay is based on the use of a recombinant yeast strain that harbours a human estrogen receptor gene ( pierrat et  al . , 1992 ) and is used to measure the estrogenic activity of environmental samples ( baker , 2001 ) .
the bacteria were grown either with manganese and ee2 or with manganese alone ( control conditions ) for 72  h ( until latestationary stage of growth ) as described above .
one millilitre of each culture was then filtered through a 0.22  mfilter and filtrates were used for the yes assay .
the yes assay was performed as described ( de boever et  al . , 2001 ) in 96well plates in which 10  l of filtrates diluted twice with dimethyl sulfoxide were incubated with 240  l of the genetically modified saccharomyces cerevisiae at an optical density of 0.25 at 610  nm .
additional plates with serial dilutions of either the medium alone ( negative control ) , or of medium supplemented with ee2 in dmso ( positive control ) were made and equally tested .
estrogenic activity responses were recorded as the absorbance at 540  nm divided by the optical density at 630  nm [ ( a540/a630)net ] ( sunrise , plate reader , tecan , grdig , austria ) . based on the data obtained , dose  response curves for doses versus
the data were fitted by a fourparametric logistic model using the marquardtlevenberg algorithm ( sigmaplot 4.0 ; spss inc . ,
chicago , il , usa ; de boever et  al . , 2001 ) .
 summarymanganese ( ii ) and manganeseoxidizing bacteria were used as an efficient biological system for the degradation of the xenoestrogen 17ethinylestradiol ( ee2 ) at trace concentrations . 
 mn2+derived higher oxidation states of mn ( mn3 + , mn4 + ) by mn2+oxidizing bacteria mediate the oxidative cleavage of the polycyclic target compound ee2 . 
 the presence of manganese ( ii ) was found to be essential for the degradation of ee2 by leptothrix discophora , pseudomonas putida mb1 , p.  putida mb6 and p.  putida mb29 . 
 mn2+dependent degradation of ee2 was found to be a slow process , which requires multifold excess of mn2 + and occurs in the late stationary phase of growth , implying a chemical process taking place . 
 ee2derived degradation products were shown to no longer exhibit undesirable estrogenic activity . 
Introduction
Results and discussion
Experimental procedures
Bacterial strains and culture conditions
Measurement of Mn oxidation
HPLC analysis
Yeast estrogen screen