btex arıtımı

882019 btex arıtımı

httpslidepdfcomreaderfullbtex-aritimi 17

BTEX biodegradation by bacteria from ef uents of petroleum re neryDacircnia Elisa Christofoletti Mazzeo a Carlos Emiacutelio Levyb Dejanira de Franceschi de Angelis c Maria Aparecida Marin-Morales a

a Department of Biology Institute of Biosciences UNESP mdashUniv Estadual Paulista mdashUNESP Av 24-A 1515 13506-900 Rio Claro SP Brazilb Department of Clinical Pathology Faculty of Medical Sciences State University of Campinas mdashUNICAMP Rua Alexander Fleming 105 13081-970 Campinas SP Brazilc Department of Biochemistry and Microbiology Institute of Biosciences UNESP mdash Univ Estadual Paulista (UNESP) Av 24-A 1515 13506-900 Rio Claro SP Brazil

a b s t r a c ta r t i c l e i n f o

Article historyReceived 1 February 2010Received in revised form 29 June 2010Accepted 1 July 2010Available online 23 July 2010

KeywordsBTEXGenotoxicityMutagenicity Allium cepaHTC cellsPseudomonas putida

Groundwater contamination with benzene toluene ethylbenzene and xylene (BTEX) has been increasingthus requiring an urgent development of methodologies that are able to remove or minimize the damagesthese compounds can cause to the environment The biodegradation process using microorganisms has beenregarded as an ef cient technology to treat places contaminated with hydrocarbons since they are able tobiotransform andor biodegrade target pollutants To prove the ef ciency of this process besides chemicalanalysis the use of biological assessments has been indicated This work identi ed and selected BTEX-biodegrading microorganisms present in ef uents from petroleum re nery and evaluated the ef ciency of microorganism biodegradation process for reducing genotoxic and mutagenic BTEX damage through twotest-systems Allium cepa and hepatoma tissue culture (HTC) cells Five different non-biodegraded BTEXconcentrations were evaluated in relation to biodegraded concentrations The biodegradation process wasperformed in a BOD Trak Apparatus (HACH) for 20 days using microorganisms pre-selected throughenrichment Although the biodegradation usually occurs by a consortium of different microorganisms theconsortium in this study was composed exclusively of ve bacteria species and the bacteria Pseudomonas putida was held responsible for the BTEX biodegradation The chemical analyses showed that BTEX wasreduced in the biodegraded concentrations The results obtained with genotoxicity assays carried out withboth A cepa and HTC cells showed that the biodegradation process was able to decrease the genotoxicdamages of BTEX By mutagenic tests we observed a decrease in damage only to the A cepa organismAlthough no decrease in mutagenicity was observed for HTC cells no increase of this effect after thebiodegradation process was observed either The application of pre-selected bacteria in biodegradationprocesses can represent a reliable and effective tool in the treatment of water contaminated with BTEXmixture Therefore the raw petroleum re nery ef uent might be a source of hydrocarbon-biodegradingmicroorganisms

copy 2010 Elsevier BV All rights reserved

1 Introduction

Monoaromatic hydrocarbons such as benzene toluene ethylben-zene and xylene (BTEX) represent an important class of environmentalcontaminants because of their recognized toxicity to different organ-isms( Anneseret al2008 Joetal2008 )Theyare widelyused chemicalsubstances in several industrial processes ( Hutchins et al 1991 Lin etal 2010 ) besides being present in high amounts in fossil fuels ( ASTDR2004 ) what determines contamination of atmosphere soil and watersAccording to Anneser et al (2008) the high solubility of BTEX in waterrepresents a serious risk of groundwater contamination

The high motility of such hydrocarbons in soil-water systems isrelated to their low octanol ndash water partition coef cient what leads toa slow soil absorption and consequently a preferential watertransport thereby favoring the contamination of water reservoirsonce they migrate fast in such medium ( Nakhla 2003 )

These compounds usually occur at trace levels in super cial watersas a result of their volatility However they can be found in moreelevated concentrations in groundwater being considered the prioritycontaminants of such resources and therefore included as compoundsto be evaluated in water analysis ( Falcoacute and Moya 2007 )

The frequency of groundwater contamination with hydrocarbonsincludingBTEX hasbeenincreasing ( Reusser et al 2002 ) demandingthe development of more ef cient methods to remove or minimizethe damages caused by these compounds

The conventional physical treatmentsbesides the highoperationalcosts remove the contaminants from the environment withoutdestroying or transforming them producing an accumulation of

Science of the Total Environment 408 (2010) 4334 ndash 4340

Correspondingauthor Departamento de BiologiaInstituto de BiociecircnciasUniversidadeEstadualPaulista(UNESP)Av 24-A 1515 CP19913506-900Rio Claro SPBrazilTel+5519 3526 4143 fax +55 19 3536 0009

E-mail address mammrcunespbr (MA Marin-Morales)

0048-9697$ ndash see front matter copy 2010 Elsevier BV All rights reserved

doi101016jscitotenv201007004

Contents lists available at ScienceDirect

Science of the Total Environment j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e s c i t o t e n v



toxic residues The biological processes known as bioremediation areregarded as a promising and clean technology particularly because of their simplicity low cost and ef cacy when compared to otheralternatives ( Alexander 1994 Bertin et al 2007 Massalha et al2007 )

The ability of BTEX degradation of certain microorganisms is knownsince 1908 when Stormer observed the capacity of the bacteria Bacillushexabovorum to grow aerobically in a medium containing toluene and

xylene The ability of natural microorganisms in the soil in BTEXdegradationwas rstdemonstrated byGray andThornton in1928Theseresearchers according to Gibson and Subramanian (1984) and Corseuiland Alvarez (1996 ) found 245 species of bacteria present in non-contaminated soil samples of them were able to degrade hydrocarbonsSince then several studies have been carried out in order to nd outef cient microorganisms for BTEX degradation so they could be used inenvironmental remediation for this mixture Among these microorgan-isms bacteria of the genus Pseudomonas are often cited such asPseudomonas putida (Ridgway et al 1990 Lee et al 1994 Otenio etal 2005 Shim et al 2005 Lin et al 2010 ) Pseudomonas uorescens(Ridgway et al 1990 Shim et al 2005 ) Pseudomonas aureofaciens (Douet al 2008 ) and Pseudomonasaeruginosa (Ridgway et al 1990 ) besidesother bacteria like Microbacterium lactuim Bacillus cereus (Dou et al2008 ) Rhodococcus rhodochrous (Deeb and Alvarez-Cohen 1999 ) andfungus Cladophialophora sp (Prenafeta-Bolduacute et al 2002 )

To be effective the product obtained in the decomposition afterbioremediation should consist of water carbonic gas and biomass(Duarte da Cunha and Leite 2000 ) However several studies havereported that microbial activity can turn non-toxic or low-toxiccompounds into potential toxic ones and in many cases the productsderived from bioremediation are recognizably carcinogenic ( Alexan-der 1999 ) such as polycyclic aromatic hydrocarbons ( Alexander etal 2002) and coal tars ( Bordelon et al 2000 Alexander et al 2002 )Therefore to evaluate the ef cacy of the bioremediation process intreating groundwater contaminated with petroleumit is suggested touse besides chemical analyses biological analyses by using bioassaysonce they provide reliable and consistent data about the actual riskscaused by the action of possible metabolites formed during bioreme-diation over organisms ( Reusser et al 2002 Plaza et al 2005 )

Although there are references which show that some organismsare able to biodegrade BTEX the purpose of this work was to identifythe microorganisms present in ef uents of petroleum re nery whichis too resistant to this compound and select those that best act on theBTEX biodegradation Since BTEX mixture is potentially toxic andbiodegradation processes may generate metabolites with differenttoxicity levels the ef ciency of biodegradation was assessed bygenotoxicity and mutagenicity assays using Allium cepa and mamma-lian cells as test-systems The identi cation and selection of moreef cient microorganisms on BTEX biodegradation can be useful inenvironmental remediation programs using this mixture

2 Material and methods

21 Selection of microorganisms through enrichment

A bacterialconsortium wasselected from a sampleof a rawef uentfrom a petroleumre nery whichwasused in theBTEX biodegradationat different concentrations

A total of 250 mL of water from Atibaia River (classi ed as a riverthat can be used for drinking after conventional treatment) and250 mL of the raw ef uent from a re nery petroleum containing96times10 6 CFUmL of bacteria were added into amber glass bottles withpartial sealing to allow gas changes In order to provide a minimumsource of energy phosphorus andnitrogen to the microorganisms weadded 05 g of sucrose05 gof caseinpeptoneand05 gof yeast extractto the suspension The inoculum was kept as 23 degCplusmn1 degC for 24 h in a

poorly-illuminated roomAfterwards200 μ L ofBTEX was added tothe

inoculum to serve as the only carbon sourceThe sample was placed ina shaker for 5 min at 30 rpm After 24 h another 100 μ L of pure BTEXwas added and the same agitation procedure was performedSimilarly 25 μ L of each pure compound was added every 48 hfollowed by agitation in a shaker for 5 min at 50 rpm for a period of 75 days Therefore a selective enrichmentof hydrocarbon-biodegrad-ing bacteria was accomplished allowing the maintenance of theculture up to its utilization A week before the biodegradation

procedure 150 μ L of pure BTEX was added every 48 h keeping thesame agitation step (this procedure was repeated four times) In theend the culture was ltered in a standard lter paper diluted in ultra-pure water at a proportion of 5 mL of culture to 100 mL of water andkept under aeration for 2 h Such material was used as the inoculum inthe biodegradation experiments

22 Concentration preparation

Five concentrations of the BTEX mixture comprising benzene(purity of 99 mdash CAS Nordm 71-43-2) toluene (purity of 99 mdash CAS Nordm108-88-3) ethylbenzene (purity of 9980 mdash CAS Nordm 100-41-4) andxylene (purity of 99 mixture of isomers mdash CAS Nordm 1330-20-7) wereprepared to carry out the bioassays

23 Biodegradation assay

We added 420 mL of each BTEX concentration1 mLof culture fromthe diluted microorganism suspensionandonepillowof BOD nutrientbuffer (Hach) into proper amber glass bottles for Biochemical OxygenDemand (BOD) assays The bottles were adapted to BOD TrakApparatus(HACH) andconnectedto a singletube attached to pressuresensors inorder to assurea completelyclosedsystem thereby avoidingchanges in external atmosphere pressure that could interfere with theexperimental data The liquid was kept under constant agitation at20plusmn1 degC for 20 days

This apparatus determines the BOD by quantifying the pressuredrop within the sealed bottles The pressure within the systemdecreases because the available oxygen is consumed by the micro-organisms being expressed as mgL The CO 2 produced by themicroorganisms through oxidation of organic matter presents noeffectson thepressure measurement once it is removed by thecrystalsof lithium hydroxide present in the bottle lids that have no contactwith the samples

A bottle containing only the microorganism culture and thenutrient buffer was also prepared to serve as a biodegradation control(white sample) To provide reliable data this process was performedtwice By the end of the process an aliquot of the sample wasseparated for quantitative chemical analysis of BTEX

24 Chemical analysis

The BTEX analysis in the tested samples prior and after the

biodegradation process was performed by the Analytical TechnologyCompany Laboratory (Satildeo Paulo mdash Brazil) The methods used in thisanalysis were injection using Headspace and gas chromatographywith photoionization detector (GCPID) according to USEPA-SW846method 8021B

25 Isolation and identi cation of microorganisms in the inoculum

The inoculum obtained from the rawef uent of petroleumre nerywas homogenized diluted in 085 M saline solution and spread ontoPetri dishes containing blood agar chocolate agar Sabouraud agarCLED agar andMacConkeyagar medium The plates were incubated ina chamber at 35 degC for 7 days Five biotypes were isolated from thecolonies in the plates and they were identi ed through Vitek IIreg

BioMerieux automatic microbial analyzing system

4335DEC Mazzeo et al Science of the Total Environment 408 (2010) 4334 ndash4340



26 Identi cation of BTEX-degrading microorganisms

In order to verify whether the identi ed microorganisms in theinoculum were able to degrade BTEX or not we used a modi edBushnell ndash Haas medium(substitution of ammonium nitrate for OXOIDLP037 bacteriologic peptone) which contains all the necessarynutrients for the bacterial growth except carbon sources Thismodi ed culture medium was separated into assay tubes A speci c

amount of BTEX (5 μ l 10 μ l 15 μ l or 20 μ l) and 100 μ l of the turbidityof each bacterial isolate in saline solution at 05 McFarland scale wasadded to each tube This procedure was repeated to each isolatedbacteria Another test was carried out by adding the bacteria poolidenti ed in the original bacterial consortium into the Bushnell ndash Haasmedium with the different BTEX concentrations mentioned aboveOne tube containing 01 glucose and BTEX-free was used as apositive control of bacterial growth P aeruginosa ATCC 27853 wasused as a positive growth control in medium with BTEX All assayswere performed in duplicate

Thetubeswere incubatedin a chamber at35 degCfor10 dayswith dailyobservations The qualitative analysis of the bacterial growth was basedon visual inspection of each tube according to the following criteria

(+) positive turbidity poor growth(++) positive turbidity moderate growth(+++) positive turbidity intense growth(minus ) negative turbidity no growth

The positive samples were replicated reisolated and to con rmthe results reidenti ed to certify the absence of any environmentalcontamination

27 Genotoxicity and mutagenicity tests

271 In vivo test The biologic material used in the present study as a vegetal test-

system to evaluate both the genotoxic and mutagenic effects of BTEX

concentrations and their biodegraded samples comprised seeds of Acepa (2n =16 chromosomes) of the same stock and cultivar ( ldquo baiaperiforme rdquo onion)

The assay was performed in glass jars which have a Te onmembrane in their lids to prevent volatilization of compounds fromthe biodegraded samples into the atmosphere

Theseeds were continuously exposed to different biodegraded BTEXconcentrations using distinct jars for each tested concentration Afterreaching 2 cm in length the roots were collected and xed in Carnoys xative 31 (ethanolacetic acid vv) The control tests were carried outwith ultra-pure water (negative control) andmethyl methanesulfonate(MMS) at a concentration of 4times10 minus 4 (positive control)

Theslidepreparation followed theproceduredescribedby Fernandeset al (2007) Ten slides were analyzed per treatment comprising 500cells perslide totalizing 5000 cells foreachselected BTEX concentrationincluding biodegraded and non-biodegraded samples

To determine the potential induction of chromosomal aberrationsall the possible chromosomal abnormalities found in the cells of eachslide per treatment were identi ed and counted The evaluation of mutagenic effects was carried out by observing and counting themicronucleated cells in all slides of each treatment

The obtained results with both non-biodegraded and degradedBTEX concentrations were compared using the Mann ndash Whitneystatistical test with a level of signi cance of 005

272 In vitro test In vitro test was performed using HTC (hepatoma tissue culture)

cells isolated from Rattus novergicus to evaluate both the genotoxicity

(comet assay) and mutagenicity (micronucleimdash

MN test) of BTEX

mixture before andafter the biodegradationprocess These cells wereobtained in the Cell Bank of Rio de Janeiro Brazil

To carry out the micronuclei test the cells were previouslycultured for a whole cell cycle (24 h) Afterwards simultaneous 24-htreatments were performed for each BTEX concentration by adding ineach tube 50 μ L of the speci c mixture (non-remediated andremediated ones) methyl methane sulfonate (MMS) (4times10 minus 2 M)(positive control) saline phosphate buffer mdash PBS (negative control)

and white (biodegradation control)Following the treatments the cells were washed twice in PBS and5 mL of complete culture medium with 3 μ gmL of cytochalasin B wasadded After 28 h the cells were harvested and xed in Carnoys xative 31 (methanolacetic acid vv) Some drops of the cell culturewere placed onto cold glass slides covered with a water layer Afterair-drying the slides were stained with 5 Giemsa for 5 min About1000 binucleated cells displaying perfect cytoplasmatic and nuclearmembranes nuclei of similar sizes non-overlapping similar stainingpattern and intensity were analyzed in each repetition comprisingtwo slides per repetition totalizing 6000 cells per treatment Thestatistical analysis of the comparison in the number of cells bearingmicronuclei of each concentration and the respective biodegradedsample was performed by using the non-parametric Mann ndash Whitneytest ( p b 005)

In comet assays the cells harvested after 24 h (whole cell cycle) ina complete culture medium from the three repetitions of alltreatments were independently treated for 24 h with each testedBTEX concentration (with and without biodegradation) and controlsubstances as previously described for MN test Following this stepthe cells were washed twice in 5 mL of PBS and trypsin-digestedusing05 mL of trypsin-EDTA 0025 for up to 2 min Afterwards theprocess was interrupted by adding 5 mL of a complete culturemedium The cells were then transferred to Falcon tubes andcentrifuged at 1000 rpm for 5 min The supernatant was discardedleaving just 05 mL of solution to resuspend the pellet

Before continuing the comet assay we performed tests of cellviability in all the treatments by mixing 20 μ L of cell suspension and20 μ L of 04 trypan blue The viable cells were counted in a Neubauerchamber considering unstainedcells as viableand theblue-stained asdead After verifying a cell viability higher than 80 an aliquot of 20 μ L of cell suspension was mixed to 120 μ L of low-melting-pointagarose (37 degC) at 05 and quickly embedded onto dry slidespreviously coated with 15 normal-melting agarose at 60 degC Theslides were covered with coverslips and kept in a refrigerator (4 degC)for 20 min After careful removal of the coverslips the slides wereembedded in cold and recently-prepared lysis solution for 1 h in therefrigerator at 4 degC

The slides were then transferred to fresh electrophoresis buffer(pH N 13) for 20 min prior electrophoresis in order to denaturize theDNA The electrophoresis was run at 4 degC for 20 min at 25 V and300 mA Afterwards the slides were neutralized in 04 M Triscomprising three baths of 5 min each air dried and xed in ethanol

for 10 min The slides were stained with 30 μ L of 1times ethidiumbromide prepared from a 10times stock solution (200 μ gmL) andcovered with coverslips

The slides were analyzed under a uorescence microscope using lter B mdash 34 (excitation λ =420 ndash 490 nM barrier λ =520 nM) 40timesenlarged About 110 nucleoids per treatment were visually classi edaccording to themigrationpattern of the fragments into the followingclasses 0 (no damage) 1 (little damage) 2 (medium damage) and 3(extensive damage) as proposed by Kobayashi et al (1995) Thedamagescore wasobtainedby multiplying thenumber of nucleoids bythe number of the corresponding score (0 1 2 and 3) To estimate apossible reduction or increase in the damages caused by biodegrada-tion in relation to the original concentration the data were compared(biodegraded and non-biodegraded concentrations) by using the

Student t -test

4336 DEC Mazzeo et al Science of the Total Environment 408 (2010) 4334 ndash4340



3 Results


The results of the chemical analyses related to the BTEXquanti cation in the tested samples are shown in Table 1 Chemicalanalyses after biodegradation showed decreased BTEX amounts in alltested concentrations Since the BOD Trak Apparatus (HACH) where

the biodegradation assay was performed is an entirely closed systemthe reduction in BTEX concentrations was exclusively caused by theconsumption of the mixture as the only carbon source for the selectedbacterial pool

32 Biodegradation

In order to evaluate the biodegradation the O 2 consumption wascontinuously measured during the entire experimental period Thisprocess carried out in the BOD Trak Apparatus (HACH) comprised 20consecutive days up to the end of the experiments Being themaximum time of reading of the BOD apparatus equal to 10 days itwas necessary to carry two consecutive treatments in the samplesThe values of oxygen consumption obtained in the rst 10 days were

recorded andthe apparatuswas re-started for10 more days The BTEXbiodegradation can be visualized in Figs 1 and 2

Based on the oxygen consumption we could infer that themicroorganisms consumed the present organic matter (BTEX)Through this analysis it was possible to estimate the instant thatthe O2 consumption begins to decline indicating the moment of experiment interruption In the present study this period comprised20 days The highest indexes of O 2 consumption were observed for

concentrations BTEX 2 and BTEX 3 mainly in the

rst ten days of thedegradation experiment After this period there was a decrease inoxygen consumption for these two concentrations ( Fig 2) Forconcentration BTEX 2 we suggest that the low consumption observedin the second phase of the experiment is a consequence of low levelsof dissolved oxygen in the sample since it hadalready been consumedin large quantities in the rst phase of the process which featured alimiting factor for thecontinuity of biodegradationIn thecaseof BTEX3 the low oxygen consumption in the second phase was due to boththe low rates of O 2 present in the bottles and the low amount of BTEXpresent inthesample due toan ef cient degradation in the rst phase(Table 1) Although BTEX 1 (highest tested concentration) had thelargest amount of available carbon source the O 2 consumption wasnot that high thereby likely to indicate that this concentration wasmore toxic to the bacteria than others The O 2 consumption levels in

Table 1Quantitative chemical analysis of BTEX in samples prior and after biodegradation

Sample Compound Initial samples ( μ gL) Biodegraded samples ( μ gL) of BTEXdecreaseConcentration DL( μ gL)a DFb IV(mL)c Concentration DL( μ gL)a DFb IV(mL)c

Dilution water B NDd 030 1 1000 ndash ndash ndash ndash ndash

T NDd

E NDd

X (mp) NDd

X (o) NDd

Total NDd

BTEX 1 B 90234400 60000 2000 1000 32225400 30000 1000 001 642870T 16869800 6370000 622402E 4040400 1480900 633477X (mp) 922200 311400 662329X (o) 252000 93000 630952Total 112318800 40480700 639591


BTEX 3 B 892295 1500 50 020 803 030 1 1000 999100T 260245 295 998866E 103985 113 998913X (mp) 23635 NDd 100X (o) 7065 NDd 100

Total 1287225 1210 999059BTEX 4 B 436 030 1 1000 347 030 1 1000 204128T 2229 162 927322E 4562 NDd 100X (mp) 982 NDd 100X (o) 341 NDd 100Total 8551 509 940475

BTEX 5 B 165 030 1 1000 NDd 030 1 1000 100T 303 NDd 100E 489 NDd 100X (mp) NDd NDd ndash

X (o) NDd NDd ndash

Total 957 NDd 100

USEPA-SW 846 method 8021Ba DL detection limitb DF dilution factorc IV initial volume in the sampled

ND non detectable




both lowest concentrations (BTEX 4 and BTEX 5) were the lowestbeing similar to that recorded for the white sample


Diverse microorganisms were found in the microbial consortiumobtained from the raw ef uent of a petroleum re nery UsingMacConkey and CLED-agar media three bacteria species wereidenti ed ( Acinetobacter lwof P putida and non-fermenting Gram-negative bacilli (NFGNB) In blood-agarand chocolate-agar two otherspecies of bacteria were identi ed ( Moraxella sp and Brevundimonasdiminuta ) (Table 2) No growth of yeast or lamentous fungi wasdetected using Sabouraud-agar

All the microorganisms found in the samples were evaluated tocerti cate whether they were capable of degrading BTEX or onlysurvived in the medium containing this mixture

The results in the identi cation of BTEX-biodegrading microorgan-isms are shown in Table 2 The bacterium P putida caused turbidity inglucose-medium and in all media with BTEX The bacteria A lwof iMoraxella sp B diminuta and NFGNB caused turbidity in the mediumcontaining only glucose butthey failedto grow in the presence of BTEXindependently on the tested concentration P aeruginosa ATCC 27853used as control caused turbidity in all tubes either in the presence of absence of BTEX The tubes containing the ve lineages of bacteria fromthe ef uent of the petroleum re nery have also caused turbidity

The replication in plates obtained from those tubes with positiveturbidity ( P putida consortium of the ve bacteria and P aeruginosa )revealedthat P putida waspresent in the replicationfromboth P putida

and bacterial consortium tubes while P aeruginosa was replicated fromthe tube with P aeruginosa

Therefore P putida proved to be the only bacterium isolated fromthe raw re nery ef uent able to grow in the presence of BTEX Thelineage P aeruginosa ATCC 27853 was ef cient as a positive control inthe tests

34 Evaluation of genotoxic and mutagenic potential

341 In vivo test The results related to the frequencies of chromosomal abnormal-

ities and micronuclei tests carried out with the test-organism A cepaare shown in Table 3 A comparison between the total amount of aberrations in the BTEX mixture and their respective biodegradedsamples revealed a signi cant reduction for the BTEX concentrations1 2 and 4 indicating that the genotoxic BTEX-induced effects weredecreased after biodegradation

A comparison between the MN frequencies in non-biodegraded

and biodegraded BTEX concentrations showeda signi

cantdecreasingforthe BTEX concentrations 1 and4 thus demonstrating a reductionof the mutagenic effects caused by BTEX after the biodegradationprocess

342 In vitro test Table 4 shows data concerning MN frequencies and damage scores

obtained from HTC cells No signi cant difference was observedbetween the MN frequencies for HTC cells exposed to ve BTEXconcentrations and their respective concentrations biodegraded

Fig 1 Mean values of O2 consumption in each BTEX concentration (mgL) by the bacteria in the solution during the rst 10 experimental days

Fig 2 Mean values of O2 consumption in each BTEX concentration (mgL) by the bacteria in the solution during the last 10 experimental days




In the comet assay a signi cant decrease of genotoxic damageswas observed for all biodegraded concentrations

4 Discussion and conclusions

An environmental contaminant acts on the indigenous biota of theecosystem eliminating or selecting microorganisms in accordance totheir sensitivity in the presence of the toxic agent Among themicroorganisms present in the contaminated site microorganismscapable of using contaminants or just resisting their toxicity can befound ( Mcnaughton et al 1999 ) The microorganisms found in soilgroundwater and super cial waters are able to break downcompounds to be used as energy source thereby eliminating themfrom contaminated environments ( Pedrozo et al 2002 )

According to Kataoka (2001) the biodegradation of organiccompounds is more ef cient when the microorganisms in theinoculum are pre-selected and thus become potentially more adaptedto target pollutants Since BTEX is a very toxic mixture tests that havepromoted the selection of microorganisms through enrichment werecarried out in this work This step was important for biodegradationsuccess because selected microorganisms adapted to BTEX mixture

Shokrollahzadeh et al (2008) used microorganisms present inactivated sludge from a petrochemical industry treatment system tobiodegrade wastewater hydrocarbon contaminated The authorsfound a population of microorganisms consisted primarily of Gram-negative bacteria such as Pseudomonas Flavobacterium Comamonas Cytophaga Sphingomonas and Acidovorax and to a smaller percentageGram-positive Bacillus These results were similar to those obtained in

this work but the aforementioned authors did not test the individualpotential of each bacterium in the hydrocarbon biodegradation

Although several authors state that biodegradation is usually drivenby a consortium of different species of microorganisms including algaebacteria fungus and protozoan ( Ghiorse and Wilson 1988 Melo andAzevedo 2008 ) we canstate that the BTEX biodegradation in this workwas due solely to bacterial actionwhich were the only microorganismsresistant to the pre-selection through enrichment

Amongst the bacteria identi ed in the inoculum P putida is shownto be the only one able to degrade BTEX According to Gibson et al(1990) P putida is known as an aromatic hydrocarbon degradercapable of using benzene toluene ethylbenzene phenol and otheraromatics as the only carbon and energy source The role of the otherbacteria found in the inoculum could not be ascertained suggestingthe necessity for further tests in order to identify their putativeparticipation in the degradation of intermediate metabolites

Similar to our ndings several reports have referred to P putida asa BTEX-degrading organism Lee et al (1994) showed that the lineageP putida TB105 modi ed in laboratory was able to metabolize amixture of benzene toluene and p-xylene without forming anyintermediate metabolite Shim et al (2005) concluded that mixedcultures of P putida and P uorescens can break down all componentsin BTEX either in aerobic or anaerobic conditions leading to acomplete mineralization of the compounds free from intermediatemetabolites Otenioet al (2005) tested the activity of the bacterium P putida CCMI 852 isolated from an ef uent treatment facility over thebiodegradation of the compounds benzene toluene and xylene bothindividually and mixed The results obtained in the isolated tests witheach one of the compounds revealed that this bacterium was able tometabolize both toluene and xylene but not benzene According tothese authors the degradation analysis of BTX mixture showed thatbesides the lack of biodegradation of benzene there was a 50decrease in the degradation rate of toluene and xylene

Based on the chemical analyses results it was possible to assertthat P putida present in the raw ef uent of a petroleum re nery andused in this assay was able to break down all the BTEX componentsthereby being regarded as an effective BTEX-degrading microorgan-ism The metabolic pathway of oxidation of these compounds by thebacteria is basedon the direct oxidation of the aromatic ring by meansof mono-oxygenases or di-oxygenases to form a catechol which issubsequently brokenby 23-dioxygenase ( Hendrickx et al 2006 ) andthe metabolites generated in this second stage are consumed by theKrebs cycle (Reineke 1998 )

The petroleum re nery raw ef uent used in the present study toobtain BTEX-degrading agents showed that this ef uent can containmicroorganisms able to break down organic compounds like BTEXcharacterizing them as an important material to be used in programs

of environmental decontamination of petroleum derivatives

Table 2Bacterial growth in the absence and presence of four BTEX concentrations in modi edBushnell ndash Haas medium

Strain Glucose 01 (μ L) BTEX (μ L3 mL)

12 5 10 15 20

Acinetobacter lwof i ++ minus minus minus minusBGN + minus minus minus minusPseudomonas putida +++ ++ + + +Moraxella sp ++ minus minus minus minusBrevundimonas diminuta +++ minus minus minus minus A lwof i BGN P putidaMoraxella sp B diminuta +++ ++ + + +Pseudomonas aeruginosa ATCC27853 +++ ++ ++ + +


Table 3Frequencies of chromosomal aberrations (CA) and micronuclei (MN) in meristematiccells of Allium cepa exposed to different BTEX concentrations prior and after thebiodegradation process

Assays CA MN

Control Negative 031plusmn014 004plusmn007Positive 159plusmn057 299plusmn101White 117plusmn048 018plusmn021

BTEX 1 Non-biodegraded 321 plusmn072 031 plusmn030Biodegraded 215 plusmn081

012plusmn014


016plusmn016BTEX 3 Non-biodegraded 183 plusmn039 008 plusmn013

Biodegraded 194 plusmn078 025 plusmn021BTEX 4 Non-biodegraded 411 plusmn109 036 plusmn049

Biodegraded 162 plusmn061 010plusmn014

BTEX 5 Non-biodegraded 161 plusmn054 020 plusmn018Biodegraded 133 plusmn042 014 plusmn013

Statistically signi cant reduction according to Mann ndash Whitney test ( p b 005)

Highly signi cant reduction according to Mannndash

Whitney statistical test ( pb

001)

Table 4Frequency of micronuclei (MN) and mean scores observed in HTC cells exposed to theselected BTEX concentrations prior and after the biodegradation process

Assays MN SCORE

Control Negative 667 plusmn163 1000 plusmn265Positive 11350plusmn1679 24433plusmn2237White 1633 plusmn 234 4033 plusmn 306

BTEX 1 Non-biodegraded 2683 plusmn 172 9167 plusmn 839Biodegraded 2717 plusmn 412 5000 plusmn 1153





Statistically signi cant reduction according to t Student test ( p b 005)




Besides decreasing the BTEX amount in the samples thebiodegradation process was also ef cient in reducing the damagesto the genetic material of both HTC and A cepa cells According toPhillips et al (2000) the bioremediation process of sites contaminat-ed with hydrocarbons must be monitored by using a battery of bioindicators that show different sensitivity levels In our studies Acepa test organism was used due to the ef ciency in diagnosinghydrocarbon genotoxic and mutagenic potentials ( Hoshina and

Marin-Morales 2009 Leme and Marin-Morales 2009 ) as well asmammalian cell culture since it is a widely used test-system to assessenvironmental contamination ( Cardozo et al 2006 )

A comparison between the results obtained from BTEX concentra-tions and their respective biodegraded concentrations showed asigni cant reduction of both genotoxic and mutagenic damages in themeristematic cells of A cepa In the case of HTC cells the biodegra-dation process was able to decrease the genotoxic damages Althoughno decrease in mutagenicity was observed for HTC cells no increase of this effect after thebiodegradationprocess wasobserved either By theobserved results these test-systems are good indicators of BTEXgenotoxicityand mutagenicityand canbe usedin effective evaluationsof this compound biodegradation

From the results obtained we observed that the biodegradationtime testedwas insuf cient forthe total elimination of BTEX present inhigh concentrations implying the need for periods exceeding 20 daysin order to achieve this process effectiveness Another importantfactor to be considered in the biodegradation process is the amount of available oxygen in the sample which should be corresponding to thephysiological needs of the degrading microorganism However weconclude that the bacteria P putida is an ef cient microorganism forBTEX biodegradation and suitable for remediation of environmentscontaminated with this compound

Acknowledgements

The authors would like to thank Prof Dr Reneacute Peter Schineiderfrom the Departamento de Microbiologia Instituto de CiecircnciasBiomeacutedicas at USP mdash Satildeo Paulo for his important contribution andsuggestions about BTEX and the Programme of Human ResourcesANPFINEPMCTCTPETRO PRH-05 at the Universidade EstadualPaulista (UNESP) Rio Claro SP Brazil for the nancial support

References

Alexander M Biodegradation and bioremediation San Diego Academic Press 1994Alexander M Biodegradation and bioremediation 2nd ed San Diego Academic Press

1999Alexander RR Tang J Alexander M Genotoxicity is unrelated to total concentration of

priority carcinogenic polycyclic aromatic hydrocarbons in soils undergoingbiological treatment J Environ Qual 200231150 ndash 4

AnneserB Einsiedl F Meckenstock RURichters L Wisotzky F GrieblerC High-resolutionmonitoring of biogeochemical gradients in a tar oil-contaminated aquifer ApplGeochem 2008231715 ndash 30

ATSDR Agency for Toxic Substances and Disease Registry Interaction pro le forbenzene toluene ethylbenzene and xylenes (BTEX) Atlanta US Department of

Health and Human Services Public Health Service Agency for Toxic SubstancesandDisease Registry 2004Bertin L Di Gioia D Barberio C Salvadori L Marchetti L Fava F Biodegradation of

polyethoxylated nonylphenols in packed-bed bio lm reactors Ind Eng Chem Res2007466681 ndash 7

Bordelon NR Donnelly KC King LC Wolf DC Reeves WR George SE Bioavailability of the genotoxiccomponentsin coaltar contaminated soilsin Fischer 344 ratsToxicolSci 20005637 ndash 48

Cardozo TR Rosa DP Feiden IR Rocha JAV Oliveira NCD Pereira TS et al Genotoxicityand toxicity assessment in urban hydrographic basins Mutat Res 200660383 ndash 96

Corseuil HX Alvarez PJJ Natural bioremediation perspective for BTX contaminatedgroundwater in Brazil effect of ethanol Water Sci Technol 199634311 ndash 8

Deeb RA Alvarez-Cohen L Temperature effects and substrate interactions during theaerobic biotransformation of BTEX mixtures by toluene-enriched consortia andRhodococcus rhodochrous Biotech Bioeng 199962526 ndash 36

Dou J Liu X Hu Z Substrate interactions during anaerobic biodegradation of BTEX bythe mixed cultures under nitrate reducing conditions J Hazard Mater 2008158264 ndash 72

Duarte da Cunha C Leite SGF Gasoline biodegradation in different soil microcosmsBraz J Microbiol 20003145 ndash 9

Falcoacute IP Moya MN Analysis of volatile organic compounds in water In Nollet LMLeditor Handbook of water analysis New York CRC Press 2007 p 599 ndash 666

Fernandes TCC Mazzeo DEC Marin-Morales MA Mechanism of micronuclei formationin polyploidizated cells of Allium cepa exposed to tri uralin herbicide PesticideBiochem Physiol 200788252 ndash 9

Ghiorse WC Wilson JL Microbial ecology of the terrestrial subsurface Adv ApplMicrobiol 198833107 ndash 72

Gibson DT Subramanian V Microbialdegradation of aromatichydrocarbons In GibsonDT editor Microbial degradation of organic compounds New York Marcel Dekker

Inc 1984 p 181ndash

252Gibson DT Zylstra GJ Chauhan S Biotransformations catalyzed by toluene dioxygenasefrom Pseudomonasputida F1InSilverS Chakrabarty AMIglewskiB KaplanS editorsPseudomonas biotransformations pathogenesis and evolving biotechnologyWashington DC American Society for Microbiology 1990 p 121 ndash 32

Hendrickx BJuncaH VosahlovaJ LindnerA Roumlegg I Bucheli-WitschelM etal Alternativeprimer sets for PCR detection of genotypes involved in bacterial aerobic BTEXdegradation distribution of the genes in BTEX degrading isolates and in subsurfacesoils of a BTEX contaminated industrial site J Microbiol Methods 200664250 ndash 65

Hoshina MM Marin-Morales MA Micronucleus and chromosome aberrations inducedin onion ( Allium cepa) by a petroleum re nery ef uent and by river water thatreceives this ef uent Ecotoxicol Environ Saf 2009722090 ndash 5

Hutchins SR Sewell GW Kovacs DA Smith GA Biodegradation of aromatichydrocarbons by aquifer microorganisms under denitrifying conditions EnvironSci Technol 19912568 ndash 76

Jo MS Rene ER Kim SH Park HS An analysis of synergistic and antagonistic behaviorduring BTEX removal in batch system using response surface methodology JHazard Mater 20081521276 ndash 84

Kataoka APAG Biodegradaccedilatildeo de resiacuteduo oleoso de re naria de petroacuteleo pormicrorganismos isolados de ldquo landfarming rdquo Rio Claro Brasil (PhDThesis mdash Institutode Biociecircncias Unesp mdash Rio Claro) 2001

Kobayashi H Sugiyama C Morikama Y Hayashi M Sofuni T A comparison betweenmanual microscopic analysis and computerized image analysis in the cell gelelectrophoresis MMS Commun 19953103 ndash 15

Lee JY Roh JR Kim HS Metabolic engineering of Pseudomonas putida for thesimultaneous biodegradation of benzene toluene and p-xylene mixture Biotech-nol Bioeng 1994431146 ndash 52

Leme DM Marin-Morales MA Allium cepa test in environmental monitoring a reviewon its application Mutat Res 200968271 ndash 81

Lin C-W Chen L-H Yet-Pole I Lai C-Y Microbial communities and biodegradation inlab-scale BTEX-contaminated groundwater remediation using an oxygen-releasingreactive barrier Bioprocess Biosyst Eng 201033383 ndash 91

Massalha N Basherr S Sabbah I Effect of adsorption and bead size of immobilizedbiomass on the rate of biodegradation of phenol at high concentration levels IndEng Chem Res 2007466820 ndash 4

Mcnaughton SJ Stephen JR Venosa AD Davis GA Chang YJ White DC Microbialpopulation changes during bioremediation of an experimental oil spill ApplEnviron Microbiol 1999653566 ndash 74

Melo IS Azevedo JL Microbiologia ambiental 2nd ed Jaguariuacutena Embrapa-CNPMA2008

Nakhla G Biokinetic modeling of in situ bioremediation of BTX compounds mdash impact of process variable and scaleup implications Water Res 2003371296 ndash 307

Otenio MH Silva MTL Marques MLO Roseiro JC Bidoia ED Benzene toluene andxylene biodegradation by Pseudomonas putida CCMI 852 Braz J Microbiol 200536258 ndash 61

Pedrozo MFM Barbosa EM Corseuil HX Schneider MR Linhares MM Ecotoxicologia eavaliaccedilatildeo de risco do petroacuteleo Salvador NEAMA 2002

Phillips TM Liu D Seech AG Lee H Trevors JT Monitoring bioremediation in creosote-contaminated soils using chemical analysis and toxicity tests J Ind MicrobiolBiotechnol 200024132 ndash 9

Plaza G Nalecz-Jawecki G Ul g K Brigmon RL Assessment of genotoxic activity of petroleum hydrocarbon-bioremediated soil Ecotoxicol Environ Saf 200562415 ndash 20

Prenafeta-Bolduacute FX Vervoort J GrotenhuisJTC vanGroenestijnJW Substrate interactionsduring the biodegradation of benzene toluene ethylbenzene and xylene (BTEX)hydrocarbons by the fungus Cladophialophora sp strain T1 Appl Environ Microbiol

2002682660ndash

5Reineke W Development of hybrid strains for the mineralization of chloroaromatics bypatchwork assembly Annu Rev Microbiol 199852287 ndash 331

Reusser DEIstok JD BellerHR Field JAInsitu transformation of deuteratedtolueneandxylene to benzylsuccinic acid analogues in BTEX-contaminated aquifers EnvironSci Technol 2002364127 ndash 34

Ridgway HF Safarik J Phipps D Carl P Clark D Identi cation and catabolic activity of well-derived gasoline-degrading bacteria from a contaminated aquifer ApplEnviron Microbiol 1990563565 ndash 75

Shim H Hwang B Lee SS Kong SH Kinetics of BTEX biodegradation by a coculture of Pseudomonas putida and Pseudomonas uorescens under hypoxic conditionsBiodegradation 200516319 ndash 27

Shokrollahzadeh S Azizmohseni F Golmohammad F Shokouhi H Khademhaghighat FBiodegradation potential and bacterial diversity of a petrochemical wastewatertreatment plant in Iran Bioresour Technol 2008996127 ndash 33




toxic residues The biological processes known as bioremediation areregarded as a promising and clean technology particularly because of their simplicity low cost and ef cacy when compared to otheralternatives ( Alexander 1994 Bertin et al 2007 Massalha et al2007 )

The ability of BTEX degradation of certain microorganisms is knownsince 1908 when Stormer observed the capacity of the bacteria Bacillushexabovorum to grow aerobically in a medium containing toluene and

xylene The ability of natural microorganisms in the soil in BTEXdegradationwas rstdemonstrated byGray andThornton in1928Theseresearchers according to Gibson and Subramanian (1984) and Corseuiland Alvarez (1996 ) found 245 species of bacteria present in non-contaminated soil samples of them were able to degrade hydrocarbonsSince then several studies have been carried out in order to nd outef cient microorganisms for BTEX degradation so they could be used inenvironmental remediation for this mixture Among these microorgan-isms bacteria of the genus Pseudomonas are often cited such asPseudomonas putida (Ridgway et al 1990 Lee et al 1994 Otenio etal 2005 Shim et al 2005 Lin et al 2010 ) Pseudomonas uorescens(Ridgway et al 1990 Shim et al 2005 ) Pseudomonas aureofaciens (Douet al 2008 ) and Pseudomonasaeruginosa (Ridgway et al 1990 ) besidesother bacteria like Microbacterium lactuim Bacillus cereus (Dou et al2008 ) Rhodococcus rhodochrous (Deeb and Alvarez-Cohen 1999 ) andfungus Cladophialophora sp (Prenafeta-Bolduacute et al 2002 )

To be effective the product obtained in the decomposition afterbioremediation should consist of water carbonic gas and biomass(Duarte da Cunha and Leite 2000 ) However several studies havereported that microbial activity can turn non-toxic or low-toxiccompounds into potential toxic ones and in many cases the productsderived from bioremediation are recognizably carcinogenic ( Alexan-der 1999 ) such as polycyclic aromatic hydrocarbons ( Alexander etal 2002) and coal tars ( Bordelon et al 2000 Alexander et al 2002 )Therefore to evaluate the ef cacy of the bioremediation process intreating groundwater contaminated with petroleumit is suggested touse besides chemical analyses biological analyses by using bioassaysonce they provide reliable and consistent data about the actual riskscaused by the action of possible metabolites formed during bioreme-diation over organisms ( Reusser et al 2002 Plaza et al 2005 )

Although there are references which show that some organismsare able to biodegrade BTEX the purpose of this work was to identifythe microorganisms present in ef uents of petroleum re nery whichis too resistant to this compound and select those that best act on theBTEX biodegradation Since BTEX mixture is potentially toxic andbiodegradation processes may generate metabolites with differenttoxicity levels the ef ciency of biodegradation was assessed bygenotoxicity and mutagenicity assays using Allium cepa and mamma-lian cells as test-systems The identi cation and selection of moreef cient microorganisms on BTEX biodegradation can be useful inenvironmental remediation programs using this mixture

2 Material and methods

21 Selection of microorganisms through enrichment

A bacterialconsortium wasselected from a sampleof a rawef uentfrom a petroleumre nery whichwasused in theBTEX biodegradationat different concentrations

A total of 250 mL of water from Atibaia River (classi ed as a riverthat can be used for drinking after conventional treatment) and250 mL of the raw ef uent from a re nery petroleum containing96times10 6 CFUmL of bacteria were added into amber glass bottles withpartial sealing to allow gas changes In order to provide a minimumsource of energy phosphorus andnitrogen to the microorganisms weadded 05 g of sucrose05 gof caseinpeptoneand05 gof yeast extractto the suspension The inoculum was kept as 23 degCplusmn1 degC for 24 h in a

poorly-illuminated roomAfterwards200 μ L ofBTEX was added tothe

inoculum to serve as the only carbon sourceThe sample was placed ina shaker for 5 min at 30 rpm After 24 h another 100 μ L of pure BTEXwas added and the same agitation procedure was performedSimilarly 25 μ L of each pure compound was added every 48 hfollowed by agitation in a shaker for 5 min at 50 rpm for a period of 75 days Therefore a selective enrichmentof hydrocarbon-biodegrad-ing bacteria was accomplished allowing the maintenance of theculture up to its utilization A week before the biodegradation

procedure 150 μ L of pure BTEX was added every 48 h keeping thesame agitation step (this procedure was repeated four times) In theend the culture was ltered in a standard lter paper diluted in ultra-pure water at a proportion of 5 mL of culture to 100 mL of water andkept under aeration for 2 h Such material was used as the inoculum inthe biodegradation experiments

22 Concentration preparation

Five concentrations of the BTEX mixture comprising benzene(purity of 99 mdash CAS Nordm 71-43-2) toluene (purity of 99 mdash CAS Nordm108-88-3) ethylbenzene (purity of 9980 mdash CAS Nordm 100-41-4) andxylene (purity of 99 mixture of isomers mdash CAS Nordm 1330-20-7) wereprepared to carry out the bioassays

23 Biodegradation assay

We added 420 mL of each BTEX concentration1 mLof culture fromthe diluted microorganism suspensionandonepillowof BOD nutrientbuffer (Hach) into proper amber glass bottles for Biochemical OxygenDemand (BOD) assays The bottles were adapted to BOD TrakApparatus(HACH) andconnectedto a singletube attached to pressuresensors inorder to assurea completelyclosedsystem thereby avoidingchanges in external atmosphere pressure that could interfere with theexperimental data The liquid was kept under constant agitation at20plusmn1 degC for 20 days

This apparatus determines the BOD by quantifying the pressuredrop within the sealed bottles The pressure within the systemdecreases because the available oxygen is consumed by the micro-organisms being expressed as mgL The CO 2 produced by themicroorganisms through oxidation of organic matter presents noeffectson thepressure measurement once it is removed by thecrystalsof lithium hydroxide present in the bottle lids that have no contactwith the samples

A bottle containing only the microorganism culture and thenutrient buffer was also prepared to serve as a biodegradation control(white sample) To provide reliable data this process was performedtwice By the end of the process an aliquot of the sample wasseparated for quantitative chemical analysis of BTEX


The BTEX analysis in the tested samples prior and after the

biodegradation process was performed by the Analytical TechnologyCompany Laboratory (Satildeo Paulo mdash Brazil) The methods used in thisanalysis were injection using Headspace and gas chromatographywith photoionization detector (GCPID) according to USEPA-SW846method 8021B

25 Isolation and identi cation of microorganisms in the inoculum

The inoculum obtained from the rawef uent of petroleumre nerywas homogenized diluted in 085 M saline solution and spread ontoPetri dishes containing blood agar chocolate agar Sabouraud agarCLED agar andMacConkeyagar medium The plates were incubated ina chamber at 35 degC for 7 days Five biotypes were isolated from thecolonies in the plates and they were identi ed through Vitek IIreg

BioMerieux automatic microbial analyzing system






















MN test) of BTEX









Student t -test




3 Results




32 Biodegradation









T NDd

E NDd

X (mp) NDd

X (o) NDd

Total NDd






X (o) NDd NDd ndash

Total 957 NDd 100


ND non detectable







































12 5 10 15 20




Assays CA MN



012plusmn014









001)


Assays MN SCORE















Acknowledgements


References


















Inc 1984 p 181ndash




















2002682660ndash



























MN test) of BTEX









Student t -test




3 Results




32 Biodegradation









T NDd

E NDd

X (mp) NDd

X (o) NDd

Total NDd






X (o) NDd NDd ndash

Total 957 NDd 100


ND non detectable







































12 5 10 15 20




Assays CA MN



012plusmn014









001)


Assays MN SCORE















Acknowledgements


References


















Inc 1984 p 181ndash




















2002682660ndash









3 Results




32 Biodegradation









T NDd

E NDd

X (mp) NDd

X (o) NDd

Total NDd






X (o) NDd NDd ndash

Total 957 NDd 100


ND non detectable







































12 5 10 15 20




Assays CA MN



012plusmn014









001)


Assays MN SCORE















Acknowledgements


References


















Inc 1984 p 181ndash




















2002682660ndash












































12 5 10 15 20




Assays CA MN



012plusmn014









001)


Assays MN SCORE















Acknowledgements


References


















Inc 1984 p 181ndash




















2002682660ndash













Acknowledgements


References


















Inc 1984 p 181ndash




















2002682660ndash







btex arıtımı

Documents