alkyl polyethoxylates ( apeo ) , widely used as industrial and domestic surfactants , are added to a variety of products such as dispersants , emulsifiers , detergents , dyes , antioxidants , pesticides , spermicides , and cosmetics [ 14 ] .
most of these compounds including nonylphenol polyethoxylates ( npeox ) are incorporated to aqueous solutions , and after being used , they are discharged in industrial or municipal water waste and eventually enter water treatment plants [ 5 , 6 ] . due to their persistency at low temperatures npeox
remain in the environment and could be bioaccumulated , which is harmful to animals , humans , and other biological aquatic species [ 710 ] , as its degradation products are more toxic than the original molecule [ 1 , 3 , 8 , 1113 ] .
the toxic effects exerted by npeox and its degradation products include reduction of spermatozoid number , increase of testicular cancer , and feminism in aquatic male species [ 7 , 8 , 14 , 15 ] .
the highly ethoxylated npeox compounds lack estrogenic activity , whereas the low ethoxylated ones , included nonylphenol , which arise from nonylphenol ethoxylates by degradation in natural environments , do affect fishes , amphibians , birds , mammalians , invertebrates species such as crustaceans , mollusks , algae , yeast , and plants [ 3 , 1619 ] .
these compounds also negatively affect microbial biomass growth by competitive inhibition mechanisms when the biomass is not acclimated .
apparently , the estrogenic activity is induced because nonylphenol mimics the structure of the female sexual hormones , the estrogens [ 5 , 21 , 22 ] .
thus , nonylphenol and ethoxylated alkylphenols have been classified as pseudoestrogens and endocrine disrupters because of the harmful effects on the endocrine systems , the reproductive cycles , and other vital functions in humans and other animal species [ 4 , 6 , 13 , 23 , 24 ] .
npeox degradation occurs through several pathways : ( i ) cleavage of the ether bonds with the generation of alkyl - phenol derivatives with shorter ethoxylated chains , which yields mono- and diethylated compounds ( npeo1 and npeo2 ) that could further be carboxylated to form ethoxyacetic and acetic nonylphenol acids [ 11 , 2527 ] .
( ii ) -carboxylation of the ethoxylated chain , yielding different polyethoxylated derivatives , being the most abundant the diethoxylated species ( npeo2c ) ; simultaneously , it is possible that the oxidation of the nonyl chain generates dicarboxylic compounds ( cnpeo1c ) , without production of nonylphenol .
( iii ) simultaneous shortening of the ethoxylated and alkylic chains to produce propyl and heptyl diethoxylated compounds   and ( iv ) oxidation of the polyethoxylated chain , without any shortening , to yield the corresponding carboxylic acids [ 27 , 28 ] .
besides , it has been reported that the initial step in the npeox degradation occurs on the ethoxylated moiety of the molecule , followed by further attack on the aromatic nonylphenol ring .  
the degradation of the aromatic ring occurs under aerobic conditions yielding several soluble metabolites which are finally degraded to co2 ; the kinetic order of the reaction corresponds to a first - order process [ 31 , 32 ] . in anaerobiosis ,
pseudomonas putida degraded npeox , with different length chains ( x = 6 , 9 and 20 ) , yielding as a final product npeo2 , no carboxylic acids were detected . 
 
sphingomonas xenophaga was able to cleave the aliphatic nonyl chain , bound to the phenol , depending on a specific branching pattern ; only the para isomer was degraded .  
finally , degradation of npeox is carried out by several bacterial genera : pseudomonas , sphingomonas , cupriavidus , ralstonia , achromobacter , staphylococcus , ochrobactrum , castellania , variovorax , bacillus , and psychrobacter [ 25 , 27 , 28 , 3335 ] . due to the negative effects of npeox on animals and the natural environment ,
its use for manufacturing domestic detergents has been banned , and their industrial applications are seriously restricted in the european community , united kingdom , united states of america , new zealand , and japan . however , in many countries , including venezuela , china , and india , legislation to control the use of such surfactants is inexistent or is not realistic .
therefore , it seems necessary to carry out studies to search for indigenous bacterial strains able to degrade npeox to be used as possible bioremediation agents on contaminated natural environments .
two of them , pseudomonas fluorescens ( strain yas2 ) and klebsiella pneumoniae ( strain yas1 ) , showed high capacity for using npeo15 as sole carbon source .
the degradation process was followed by viscometry of the previously used culturing media and measurement of the chemical and biological oxygen demands . to our knowledge , this is the first report of the npeox degrading activity by klebsiella sp .
the bacterial strains were isolated from soil samples collected in the gardens of the centro de investigaciones microbiolgicas aplicadas ( cima - uc ) , campus brbula , carabobo , venezuela . to our knowledge
the bacteria were grown in a minimal mineral medium ( mm ) containing the followings salts ( p / v ) : 1% cuso4 , 0.1  ml ; 0.5% feso4 , 2  ml ; 1% mgso4 , 0.2  ml ; 1% znso4 , 0.5  ml ; 0.7% nacl , 0.05  ml ; 0.1% nh4cl , 1.0  ml ; 0.8% nh4no3 , 0.125  ml and 1% caco3 , 0.5  ml , volume was adjusted to 1.0 l with 0.1  m phosphate buffer at appropriated ph values , as it will be indicated further ( mm medium ) .
all salts were proanalysis quality and the ethoxylated nonylphenol ( npeo15 , mw 880  g  mol ) was kindly donated by palma products , ca valencia , venezuela .
soil samples ( 100  g ) were suspended in mm medium ( 250  ml ) in 500  ml flasks , at ph 7.0 , in aerobiosis , and appropriate volumes of npeo15 were added to reach final concentrations of 3% v / v and 30% v / v ( 0.0365 and 0.365  m , resp . ) .
the systems were incubated at room temperature ( 2225c ) under constant shaking during 45 days .
aliquots ( 3  ml ) , taken at different cultivation times , seeded in nutritive broth tubes and the bacterial growth was evaluated , after 24  h at 37c , by single visual inspection of turbidity . after streaking of the total bacterial population on nutritive agar plates , after 2448  h at 37c
, colonies were selected and assessed for growth in mcconkey , kliger 's iron and oxidase media and finally the isolated colonies were stored in nutritive broth at 4c until further use .
the taxonomic identification was carried out by using the analytical profile index ( api ) 20e and 20ne systems ( biomrieux sa , france ) .
the selected bacterial colonies were grown in 250  ml flasks containing mm/3% npeo15 medium ( 50  ml ) , ph 7 , in aerobiosis for 12  h at 27c , and then aliquots were submitted to different experimental protocols.to assess the purity and viability of the colonies , aliquots ( 1  ml ) were added to the same medium ( 150  ml ) at 27c and the bacterial growth was assessed , at intervals of 3  h , over a period of 9  h , by measuring the absorption at 660  nm on a spectronic genesys ii spectrophotometer .
simultaneously , aliquots ( 1  ml ) were used , to prepare solutions by successive dilution that allowed determining the number of colony forming units per milliliter ( cfu  ml ) .
the mm/3% npeo15 medium was adjusted to different ph values ( 7 , 8 , 8.5 and 9 ) and the bacterial growth was followed as indicated in protocol i. the mm medium was adjusted to 1 , 3 , and 5% npeo15 concentrations ( 0.0121 , 0.0365 , and 0.0605  m , resp . ) .
the bacterial growth was followed as indicated in protocol i. to assess the purity and viability of the colonies , aliquots ( 1  ml ) were added to the same medium ( 150  ml ) at 27c and the bacterial growth was assessed , at intervals of 3  h , over a period of 9  h , by measuring the absorption at 660  nm on a spectronic genesys ii spectrophotometer .
simultaneously , aliquots ( 1  ml ) were used , to prepare solutions by successive dilution that allowed determining the number of colony forming units per milliliter ( cfu  ml ) .  
the mm/3% npeo15 medium was adjusted to different ph values ( 7 , 8 , 8.5 and 9 ) and the bacterial growth was followed as indicated in protocol i.   the mm medium was adjusted to 1 , 3 , and 5% npeo15 concentrations ( 0.0121 , 0.0365 , and 0.0605  m , resp . ) .
the bacterial growth was followed as indicated in protocol i. the assays were executed according to the open reflux method 5220b .
bacterial strains were incubated for 24  h in mm/1% npeo15 medium , 27c , and ph 8 , and bacterial growth was followed every 3  h , at 660  nm absorbance .
aliquots were withdrawn after 0 , 6 , and 24  h incubations , and the samples were filtered through millipore membranes ( 0.45  m pore size ) to obtain free bacterial filtrates ( fbf ) and submitted to analysis . a blank system ( without bacterial inoculums )
after 24  h at 27c , ph 8 in mm/1% npeo15 medium , inoculums ( 1 ml ) were incubated in the same medium and conditions .
aliquots were withdrawn after 6 , 24 , and 48  h incubation times and dilutions prepared in water ( 300  ml ) were incubated for 5 days at 20c in a dark and dry chamber .
volumes ( 5  ml ) of bacterial cultures grown in mm/5% npeo15 medium , ph 8 , at 27c for 12  h , were transferred to the same medium ( 300  ml ) and aliquots were withdrawn at different times for one week at 25c . immediately after the removal
, the samples were filtered through millipore membranes ( 0.45  m pore size ) to obtain the fbf and stored at 20c until further use .
the viscosity changes were determined in a cannon 50w404 ostwalt viscometer at 25c and densities in a 10  ml pycnometer .
a calibration curve relating flow times ( seconds ) in the viscometer to npeo15 concentrations ( mm ) was obtained in order to determine the remaining npeo15 concentrations .
values , at 25c , for water density ( 0.99704  g  ml ) and viscosity ( 0.8904  g  cm  s , centipoises ) were taken from weast .
relative viscosity values were calculated by the expression s = otss / too , where zero subscript refers to water and s to npeo15 aqueous solutions [ 39 , 40 ] .
from the soil samples five indigenous bacterial colonies were isolated , of which strain h grew on mm/3% npeo15 medium and the other four ( strains f , k , n , and o ) on mm/30% npeo15 .  
table 1 shows the morphological and biochemical characteristics of the isolated strains .   the bacterial growth on mm/3% npeo15 medium , ph 7 , 27c is shown in figure 1 .
strains k and o showed the highest growth rates ; both grew without lag phase and the logarithmic phase was observed until 6  h incubation time . because of this behavior both strains were chosen to perform subsequent experiments .
according to the api identification system strains k and o were identified as pseudomonas fluorescens and klebsiella pneumonia and were named yas2 and yas1 , respectively .
the behavior of the bacterial strains at different ph values is described in figure 2 .
maximal growth rates were achieved at ph 8 ( 0.04 and 0.033 absorbance units ) , whereas the minimal growth was observed at ph 9 ( 0.008 and 0.01  au ) after 6  h culturing time .
these data correlated well with the cfu ml numbers at 6  h culture : 1010 and 1010  cfu  ml at ph 8 and 9 , respectively ( data not shown ) . in figure 3 the bacterial growth patterns with respect to the npeo15 concentration in the media are shown .
maximal ( 0.09 and 0.062  au ) and minimal ( 0.019 and 0.012  au ) growth values were achieved at 5% and 1% npeo15 , respectively , at 9  h incubation times .  
usually , in the culture media of 1% and 3% the maximal growth was observed at 6  h incubation , and then it reached the stationary growth phase at 9  h. the logarithmic growth phase persisted until 9  h for the 5% npeo15 system , and then the stationary phase disappeared .
finally , a mixture culture of yas1/yas2 in 1% npeo15 medium , ph 8 , showed a synergistic effect on the bacterial growth , reaching maximal values ( 0.095  au ) at 9  h incubations and 27c , which represents a stimulatory average factor close to 5.7 with respect to the individual bacterial cultures at 1% npeo15 medium ( 0.014 and 0.02  au ) ; additionally , the lag phase was absent ( see figure 4 ) .
this stimulatory effect is probably due to npeox cometabolism by the yas1/yas2 system , as it has been described for other bacterial consortiums .
the npeo15 biodegradation was determined in fbf by following the flow time in a viscometer and by the chemical oxygen demand , also the biological oxygen demand was evaluated .  
figure 5 shows the calibration curve that relates the molarity of npeo15 solutions and their flow times in a viscometer .
the data fit the equation y = 226.9 + 0.9418x  ( r = 0.94 ) calculated by linear regression .  
tables 2 and 3 show the viscosity and density changes , flow times ( s ) , molarity ( m ) of remaining npeo15 , and density ( g  l ) and viscosity ( g  cm  s ) of the fbf corresponding to different incubation times for both bacterial strains .
a decrease in the fbf viscosity and density correlated well to the observed decrease of the determined flow times . from an initial value of 1.150  g  cm  s the viscosity was reduced to 0.8959 and 0.8490 for yas1 and yas2 systems , respectively .
these facts indicated the cleavage of the npeo15 to lower molecular weight species . on basis of these data yas1 and yas2 degraded 0.0392 ( 0.06050.0213 ) and 0.0383 npeo15 moles l ( 0.06050.0216 ) in 54.5 and 134  h , respectively .
the extension of the degradation process has been reported as temperature dependent [ 6 , 42 ] . in this study degradation
was close to 65% at 27c ; similar values have been reported by other authors [ 6 , 40 , 41 , 43 ] .  
it was also observed that foam appearing at the beginning of the incubations decreased at late incubation times . at 71  h ( yas1 ) and 163  h ( yas2 ) incubations , foam had totally disappeared and simultaneously , viscosity increases were evident in both cultures .
it is well known that several bacterial genera are able to produce viscous polymers ( mucopolysaccharides , dextrans , proteins , poly--hydroxybutyrate , polyphosphates , and xanthans ) as strategies to retain nutrients and water , as energy reserves and for defense purposes [ 4448 ] .
another aspect for consideration is the observed viscosity changes with the incubation times ; yas1 decreased the viscosity from 1.15  g  cm  s to 0.8959 in 54.5  h , a relative short time ; at 8.5  h of incubation the viscosity fell , but it suddenly rose ( 22.5  h ) to fall again at 32.5  h incubation .
this apparent data dispersion could be due to the well - known mucogenic properties of the klebsiella genus [ 44 , 46 , 47 ] , which was also observed in this study ( see table 1 ) .
on the other hand , yas2 changed the viscosity from 1.15 to 0.849  g  cm  s , in 134  h , a relative longer time , without abrupt changes .
thus , yas1 produced viscous materials at early and late incubation times , whereas yas2 did it only at late times .
synthesis of dextrans and alginates by klebsiella and pseudomonas , respectively , has been reported [ 4951 ] ; therefore , viscometry studies to evaluate degradation of viscous substances seem to be inappropriate when the bacteria are capable of synthesizing viscous polymers during the whole incubation time , such as klebsiella did .
however , if the bacteria yield viscous molecules at relatively late times during the incubation , then the viscosity changes could be a useful , cheap , and rapid method to detect biodegradation of viscous xenobiotic polymers , as in the pseudomonas system . regardless of whether the bacteria degrade npeo15 or
the bacterial polymers , differences in degradation times displayed by the bacterial strains would imply that yas1 cleaved chemical bonds near the aromatic ring in the npeo15 molecule , producing low molecular weight species and causing a rapid change in the viscosity , whereas yas2 seems to exert its action progressively on bonds near to the hydroxylated end of the surfactant ethoxylated chain , thus the decrease of the molecular mass was not as abrupt and the viscosity decrease should be slow .
the reported data in tables 2 and 3 allowed obtaining kinetic information about the npeo15 biodegradation . taking the initial ( co ) and remaining ( cx ) npeo15 concentrations at different incubation times it was possible to determine the kinetic order for the npeo15 biodegradation according to first- and second - order kinetic equations for a chemical reaction , also the kinetic rate constant k and the half life time  for the npeo biotransformation were calculated .  
 figure 6(b )
shows the ln(co / cx ) versus t plot corresponding to the yas2 data ( table 3 ) . the obtained straight line ( y = 0.0072x0.0053 ,  r = 0.9728 )
indicated the npeo15 biodegradation obeyed first - order reaction kinetics ; data from other laboratories indicated that npeo1 and npeo2 also obeyed the same order kinetics . a similar analysis for the yas1 system ( figure 6(a ) ) did not allow assigning any reaction order because of the viscous material synthesized at early and late time incubations by the yas1 strain .
the estimated values of the rate constant k and time  for yas2 were 0.0072  h and 96.3  h , respectively . for these calculations
the corresponding data at 163  h were not considered because the observed viscosity increments were probably due to the synthesis of viscous bacterial polymers and not due to npeo15 present in the media .
although the maximal bacterial growth was obtained at 5% npeo15 , the following experiments were executed at 1% npeo15 because the principal aim of this study was to propose a satisfactory solution to the real environmental npeo contamination which according to several reports should be less than 1  mg l [ 23 , 5255 ] .
the chemical and biological oxygen demands , determined in broths previously used by microorganisms , are indirect measurements of the carbonaceous substrate degradation by a microbial population .
tables 4 and 5 shows the chemical oxygen demand ( cod ) and the biological oxygen demand ( bod ) of both bacterial cultures in mm/1% npeo15 medium , ph 8 at different incubation times .   after 24  h incubation , the cod values decreased from an initial value of 20,230 mgo2 l ( zero time ) to 666 and 3,066  mgo2  l , which represent 96 and 85% npeo15 degradation for yas1 and yas2 strains , respectively ( see table 4 ) .
these low cod values , representing 4 and 15% of the initial values , indicate that both bacterial strains can probably degrade npeo15 and its low ethoxylated derivatives , including nonylphenol , as it has been reported for other bacterial strains [ 6 , 26 , 43 , 56 ] .
the bod5 data ( table 5 ) indicated that both bacterial strains consumed 99% ( 60.11/8,675 and 67.8/14,000 ) and 99.9% ( 5.26/8,675 and 5.11/14,000 ) of the available oxygen in 24 and 48  h , respectively , which implies that the npeo15 derivatives did not exert appreciable toxic effects on the bacterial strains and are biodegradable .  
npeox and its derivative degrading bacterial strains have been isolated from several natural environments and wastewater treatment plants [ 1 , 5 , 21 , 30 , 5759 ] . in this paper
the reported data indicated that indigenous bacterial strains , isolated from soil , are able to use npeo15 as the sole carbon source .
although the viscosity descent is indicative of bond cleavages in the npeo15 molecule , it does not imply the use of the degradation products for sustaining the bacterial viability .
however , the changes in the chemical and biological oxygen demands and the increments ( viability ) in the cfu  ml number in npeo15 complemented media indicate that yas1 and yas2 must use the npeo15 degradation products to satisfy their metabolic requirements and support cellular division .
p. fluorescens ( yas1 ) and k. pneumoniae ( yas2 ) thus appear to be useful biotechnological tools to bioremediate npeo contaminated waters and soils .
five bacterial strains , isolated from soil , grew on a minimal mineral medium supplemented with npeo15 ( 0.0365  m and 0.365  m ) as the sole carbon source , being pseudomonas fluorescens and klebsiella pneumoniae the two most efficient npeo15 degrading strains .
the extent of npeo15 degradation after 2448  h incubations , evaluated by cod and bod5 assays , was , 8595% and 99.9% respectively .
the kinetic rate constant ( k ) and the half life time (  ) for the npeo15 biotransformation by p. fluorescence were estimated to be 0.0072  h and 96.3  h , respectively , and the process followed first - order kinetics .
 polyethoxylated nonylphenols , with different ethoxylation degrees ( npeox ) , are incorporated into many commercial and industrial products such as detergents , domestic disinfectants , emulsifiers , cosmetics , and pesticides . 
 however , the toxic effects exerted by their degradation products , which are persistent in natural environments , have been demonstrated in several animal and invertebrate aquatic species . 
 therefore , it seems appropriate to look for indigenous bacteria capable of degrading native npeox and its derivatives . in this paper , 
 the isolation of five bacterial strains , capable of using npeo15 , as unique carbon source , is described . 
 the most efficient npeo15 degrader bacterial strains were identified as pseudomonas fluorescens ( strain yas2 ) and klebsiella pneumoniae ( strain yas1 ) . 
 maximal growth rates were reached at ph 8 , 27c in a 5% npeo15 medium . 
 the npeo15 degradation extension , followed by viscometry assays , reached 65% after 54.5  h and 134  h incubation times , while the cod values decreased by 95% and 85% after 24  h for the yas1 and yas2 systems , respectively . 
 the bod was reduced by 99% and 99.9% levels in 24  h and 48  h incubations . 
 the viscosity data indicated that the npeo15 biodegradation by yas2 follows first - order kinetics . 
 kinetic rate constant ( k ) and half life time (  ) for this biotransformation were estimated to be 0.0072  h1 and 96.3  h , respectively . 