Study on biodegradation of Mazut by newly isolated strain Enterobacter cloacae BBRC10061: improving and kinetic investigation.

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by  Khorasani, Alireza Chackoshian

Khorasanietal.IranianJournalofEnvironmentalHealthSciences&Engineering2013,10:2
IRANIAN JOURNAL OF
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ENVIRONMENTAL HEALTH
SCIENCE & ENGINEERING
RESEARCH ARTICLE Open Access
Study on biodegradation of Mazut by newly
isolated strain Enterobacter cloacae BBRC10061:
improving and kinetic investigation
Alireza Chackoshian Khorasani1, Mansour Mashreghi2* and Soheyla Yaghmaei1
Abstract
Mazut as a source content of various hydrocarbons is hard to be degraded and itscracking could turn mazut into
useful materials. Nevertheless degradation of mazut by routine methods is too expensive but applicationof
indigenous microorganisms as biocatalysts could be effective and important to lower thecosts and expand its
consumption. Mazut biodegradationcan be improved using various strategies; Therefore inthis study newly
isolated strain Enterobacter cloacaeBBRC 10061 was used ina method ofgradual addition ofmazut into medium
and itsresults were compared withsimpleaddition method. Toinvestigate degradation of mazut byBBRC 10061,
influence of increase ofmazut concentration was assayed based ongradual addition method. Alsodifferent kinetic
models were used to evaluate kinetics of theprocess. Resultsshowed that gradual addition method has been a
beneficial technique for improvement ofmazut degradation because bacterial induction to produce biosurfactant
and essential enzymes for cracking mazut was higherduring process. Althoughadditionof more mazut increased
therate ofbiodegradation but percentage of degradation decreased. pH of medium decreased during
biodegradation period whileelectric potentialincreased.Also the biodegradation kinetics was not fitted with the
biokinetic models; therefore kinetics of biodegradation ofmazut has to be studied by new models.
Keywords: Biodegradation, Mazut, Gradual addition, Biokinetic model, Enterobacter cloacae
Introduction environments is more effective, powerful and economical
Endproductofcrudeoildistillationisaresidualcompound providentthanphysicalandchemicalways[7].Assessment
namedmazutthatthepresenceofheavyhydrocarboncom- ofeffectiveparametersandconditionsonbiodegradationis
poundssuchasasphaltene,resins,longchainalkanes,cyclic verysignificanttodesignbioremediationsystem[8].Dueto
alkanes and multi cyclic aromatics in it turn mazut into a material and process, microorganism can be isolated from
high viscous and hard degradable product; On the other environments which their conditions prepare microorgan-
hand, sulphuric compounds and heavy metals in mazut ismtoturnmaterialondemand.Isolatingfromindigenous
makeuseofitmoredifficult.Mazuthasvarioustypesbased area is usuallyadvantageousto achievehigh microbial per-
on hydrocarbon compounds which usually depended on formance; moreover preparation of appropriate conditions
geographical area and storage sources which crude oil is for microorganism can help procedure because micro-
prepared from [1]. Bioprocess is suggested as effective and organism is adapted by them [9]. Various studies have
economical provident ways. Its advantage against other alreadybeenreportedaboutdetectionandisolationofable
physicalandchemicalprocessesissimplicityandlowercost microorganisms to degrade oil compounds but there are
[2-5]. What makes biodegradation as a most important notenoughreportsofbiodegradationofmazutbymicroor-
process is use of newly isolated indigenous microorga- ganisms in Iran. Indigenous microorganisms such as
nisms [6]. Biodegradation of oil derivatives contaminating Pseudomonas aeruginosa, Pseudomonas stutzeri, Provede-
nicastuartii,Acinetobactersp,BacillusCereus,Micrococcus
sp, Enterobacter cloacae and Serratia sp isolated from dif-
*Correspondence:[email protected]
2CellandMolecularResearchGroup,InstituteofBiotechnology,Ferdowsi ferent locations in Iran which were contaminated by oil
UniversityofMashhad,Mashhad,Iran compounds,suchassoilandwatersources,wereemployed
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Khorasanietal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Khorasanietal.IranianJournalofEnvironmentalHealthSciences&Engineering2013,10:2 Page2of7
http://www.ijehse.com/content/10/1/2
to degrade various hydrocarbons but not mazut [10-15]. Analyticalmethods
Biokinetic models are mathematical tools to predict and For estimation of mazut concentration, spectrophoto-
model activity and behavior in a biosystem so that can in- meter (S2000 UV/VIS) was used. To extract mazut from
vestigatedifferentkineticparametersandratecurvesbased medium, chloroformwasadded into mediumproportion-
on real and controlled data [16,17]. Therefore enough atelywithinitialconcentrationofmazut(3mlchloroform
knowledgeofmicrobialgrowthanddegradationkineticsof for < 2000 ppm, 6 mL for < 10000 ppm and 9 mL for <
matters are necessary to model biokinetics [18]. Each of 50000 ppm) and centrifuge tube was shaken and blended
simple biokinetic models already been suggested is usually till all mazut was dissolved in chloroform. Then organic
adaptedtospecialconditionsandsuitabletoparticularpro- phase content of mazut and chloroform was under and
cesses. Among models, Monod model (1949) is simplest mineral phase was up. To extract organic phase, mineral
model already been reported and other models such as partvoidedofmazutandanotherorganiccomponentsdis-
Mosermodel(1958),Webbmodel(1963),Aibaetal.model solved in chloroform was decanted. After that, organic
(1968), Haldane model (1968), Yano and Koga (1969), phase was sampled and diluted by chloroform solvent.
Teissiermodel(1970)andLevenspielmodel(1980)describ- Now absorbance of mazut in medium was measured at
ing kinetic behavior of complex biosystems are advanced 450 nm by spectrophotometer. Sample concentration was
[19,20]. measuredbasedonabsorbance-concentrationcurve.
Objectives of this study were investigation of effect of To estimate bacteria amount in medium, after sam-
gradual addition of mazut on biodegradation of hydro- pling from medium bacterial density was measured at
carbon derivative in mazut using an indigenous bacte- 600 nm by spectrophotometer and recorded as optical
rium Enterobacter cloacae BBRC 10061 isolated from oil density (OD ).
600
contaminated soil in Mashhad (Iran), and modulation of Electric potential and pH were measured by pH meter
itsbiodegradationkinetics. (3020model,Jenway).
Materials and methods Biokineticmodels
Microorganism Results of degraded mazut in this study were fitted on
E. cloacae BBRC 10061, named NO4 in this study, is in- various biokinetic models which are presented in Table 1
digenous bacteria strain isolated from oil contaminated where r is rate of mazut degradation (mg/ L /day), r
s s max
soil where buses were fixed and fuelled in Mashhad,
Table1Biokineticmodelsusedforbiodegradationofmazut
Iran. It was procured from the biochemical and bioenvi-
Model Equation
ronmental research center (BBRC) that was a local cul-
ture collection in Sharif University of Technology; Monod rs¼rsmaxKsS¼S
Tehran, Iran. The microorganism was maintained in gly- (cid:1) (cid:3)
cerolstockat−20°Cfor further use. Teissier rs¼rsmax (cid:1)e(cid:2)KSi(cid:3)(cid:2)e(cid:2)KSs
S e(cid:2)KSi
Culturemediumandexperimentalcondition Aibaetal. rs¼rsmax KsþS
The medium culture had the following composition per
l0i.t0e1r:, (MNHgSO)4S.O7H25Og,0.K1Hg,POCaC2l.52 g0,.0K1 HgP, OFeS2O.54.7gH, s2eOa YanoandKoga rs¼rsmaxKsþSþSKS21þKS322
4 2 4 2 4 2 4
salt 0.5 g, glucose 4 g and tween80 1 g. The medium Haldane(Andrews) rs¼rsmaxKsþSSþKS21
was autoclaved at 121°C for 20 min and after cooling,
sterile medium was distributed in 50 ml sterile centri- Webb rs¼rsmaxKSsðþ1SþþKSKiSÞ21
fuge tubes which were inoculated with NO4 bacteria
amountedto0.032OD600. Levenspiel rs¼rsmaxSðK1sþþSKSiÞ
Two types of culture were used, simple and gradual.
To prepare simple culture, 2000 ppm (0.1 g per 50 mL) Moser rs¼rsmax1þKs1ðS(cid:2)λÞ
mazut was added into 50 ml medium culture in centri-
fuge tube. Gradual culture was prepared at concentra- Contois rs¼rsmaxβxSþS
tions of 2000 ppm, 10000 ppm and 50000 ppm of
mazut. Every 2 day, 400 ppm, 2000 ppm and 10000 ppm Firstorder rs=aS+b
mazut were added in centrifuge tubes containing 50 ml
medium cultures to prepare gradual cultures in concen- Secondorder rs=aS2+bS+c
trationsof2000ppm,10000ppmand50000ppm.
Allcultivationswerethenincubatedfor10daysat33°C Thirdorder rs=aS2+bS2+cS+d
andinitialpH6.8withaspeedof160rpm.
Khorasanietal.IranianJournalofEnvironmentalHealthSciences&Engineering2013,10:2 Page3of7
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is maximum of mazut degradation rate (mg/ L/ day), S is Biodegradation rate was steady but bacterial growth rate
mazut concentration (ppm), x is microbial optical density fluctuated. Bacterial growth increased quickly at process
(dimensionless), K is half-saturation constant of mazut beginning and with time lapse rate decreased although
s
(mg/ L), K isinhibition constant (mg/L), K isinhibition bacteria did not enter into death phase. According to
i 1
constant (mg/ L), K is inhibition constant (mg/ L), λ is Figure 2A efficiency of degradation of mazut was very
2
constant of Moser model (dimensionless), β is constant low until fourth day but after that degradation rate be-
of Contois model (mg/ L), A (mg/ L /day), B (day), C came steady. During ten days from 2000 ppm mazut
(mg /L/ day) and D (mg/ L /day) are constants of linear added into medium just 1200 ppm remained indicating
models. Experimental data for the degraded mazut and to about %40 mazut degradation. NO4 bacterial growth
the bacteria were used as the experimental data to vali- at first four days attained the most amounts and after
date the models. The mathematical models were solved then bacteria entered into stationary phase. Growth rate
numerically in Microsoft Office Excel 2007 using the increased with degradation of simple hydrocarbons but
SOLVER tool. The kinetic parameters and constants for reduced afterdeploymentanddegradationrate remained
each timepoint were calculatedbased on themazutcon- unchanged because cracked hydrocarbons turned into
centration. A linear regression between the experimen- more digestible derivatives and bacteria grew by con-
tal data and the predicted data were computed for all suming them whereas main molecules of mazut were
the assays. degraded at foregoing rate and it was approximately in-
dependent of biomass change. Gradual addition caused
Results biosystem to be slowly adapted and this method in ge-
Influenceofgradualadditionofmazut neral could be more effective than abrupt addition at
Degradation commenced after adding 2000 ppm mazut start; because abrupt addition of mazut may cause nutri-
into medium and during the process NO4 bacteria mental shock and microbial dead resulting decrease of
degraded mazut about 1400 ppm (Figure 1A). growth rate. Bacterialgrowth wasvery fastinfirst2days
Figure1(A):Biodegradationofmazutandbacterialgrowthinsimplemedium(B)pHandelectricpotentialchangeduringmazut
biodegradationinsimplemedium.
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Figure2(A):Biodegradationofmazutandbacterialgrowthingradualmediumat400ppmadditionalconcentration.(B)pHandelectric
potentialchangeduringmazutbiodegradationingradualmediumat400ppmadditionalconcentration.(C)Biodegradationofmazutandbacterial
growthingradualmediumat2000ppmadditionalconcentration.(D)pHandelectricpotentialchangeduringmazutbiodegradationingradual
mediumat2000ppmadditionalconcentration.(E)Biodegradationofmazutandbacterialgrowthingradualmediumat10000ppmadditional
concentration.(F)pHandelectricpotentialchangeduringmazutbiodegradationingradualmediumat10000ppmadditionalconcentration..
while the rate of mazut degradation was very low microbial growth rate in first days but at following
(Figure 2A). These changes showed stage of adaptivity of process pH change was very low. The reason was that
microorganism to culture medium. Bacteria grew by acidy condition decreased bacterial activity and produce
consuming glucose in medium and were ready to de- of secondary products. Complications of production and
grade mazut. Then bacterial growth did not change and consumption of different materials in system caused
mazut was degraded in stationary phase. This status was many changes in electric potential and conductivity in
better than status which mazut degradation occurred culture medium which were difficulty legitimized; but
during microbial growth because during process, totally during the biodegradation of mazut pH reduced
amount of biomass as catalyst was approximately fixed andelectric potentialincreasedinmedium.
constant. Also this amount was equivalent to final
amount of biomass at simple method. Thus it caused Influenceofmazutconcentration
degradation percentage became higher in longer period For assessment of effectiveness of gradual method, influ-
of time with more microorganisms presented. Bacterial enceofincreaseinmazutconcentrationonbiodegradation
growth was accompanied by secondary acidy products rate was studied by this method. With increasing add-
and medium turned to acidy quickly by increase of itional concentration of about 2000 ppm in which during
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ten days 10000 ppm was in medium, mazut degradation results showed that simple process did not correlate with
percentage attained to %30. At this time residual mazut any models but gradual process at 2000 ppm additional
got 7000 ppm and biomass with less fluctuated growth concentration correlated with second and third order
gained about 3 OD (Figure 2C). Gradual addition for models. Other gradual processes data were not fitted on
600
every stage was 2000 ppm but degradation time of 2 days the models. Results also showed that given biokinetic
was very short. Only 3000 ppm was degraded at ten days models were more suitable to model gradual processes.
and bacterial growth was slow. Bacterial growth and Although in general mazut biodegradation data was not
mazut degradation were possible during 10 days due to proper to be modeled by given models but second and
mazut remained at every stage and NO4 bacteria did not third order models were more correlated than other
enter into stationary phase. Increase in concentration of models.
mazut caused inducing bacteria to degrade it and finally
more concentration was degraded. Percentage of mazut Discussion
degradation by NO4 bacteria attained to %10 when con- There are just a few studies on biodegradation of mazut
centrationofmazutinbiodegradationsystemincreasedto compared with other materials such as crude oil and
50000 ppm (Figure 2E). Maximum bacterial growth was other petroleum compounds. Therefore this project has
observedatfirst2daysandwithenteringintothestation- been anewresearch and investigation in the field of pet-
aryphasebiodegradationprocesswascarriedoutthatfinal roleum biotechnology. New isolated strain of bacterium
biomassgainedabout2OD .Infirstdaysofincubation, E. cloacae in this study was capable to biodegrade mazut
600
pHreducedfrom6.8to6relatingtoincreaseinmicrobial efficiently and was of first bacterial strain cracking
growthwhichmademediumacidy(Figure2D,F).Electric mazut. More researches on mazut biodegradation have
potentialincreased at the beginning of incubation periods already been carried out in soil medium whereas this
but when microbial growth approached stationary phase, study was completely investigated in controlled specific
changes in pH and electric potential was not observed. water condition. Bioremediation of mazut-polluted soil
ThereasonforpHreductionorincreaseinelectricalcon- by fungi species reduced 300 mg of initial concentration
ductivity of medium could be due to increase of produc- which was 5000 mg per kg soil during ten days and fur-
tionandconsumptionofdifferentmaterialswhicharethe therincubationupto140 daysreducedmazutconcentra-
resultsofincreaseinbiomassandrateofdegradation. tion down to 1500 mg per kg soil [21]. Among fungus
studied, species Trichoderma harzianum had highest
Evaluationofbiokineticmodels ability for degradation of mazut [22]. Results obtained
Biodegradation of mazut at different concentrations using from biodegradation of mazut in soil using ex situ
simple and gradual medium was fitted on the biokinetic method showed that during 50 days 3 g of 5 g mazut in
models which their results are presented in Table 2. The 1 kg soil was degraded and finally after 150 days got
Table2Linearcorrelationcoefficientsforsimpleandgradualmediums
BiokineticModel Simplemedium Gradualmedium Gradualmedium Gradualmedium
(400ppmadditional (2000ppmadditional (10000ppmadditional
concentration) concentration) concentration)
R2 R2 R2 R2
Monod 0.07 0.57 0.52 0.56
Teissier 0.06 0.57 0.52 0.56
Aibaetal. 0.16 0.57 0.52 0.56
YanoandKoga 0.1 0.60 0.52 0.56
Haldane(Andrews) 0.16 0.57 0.52 0.56
Webb 0.12 0.63 0.50 0.63
Levenspiel 0.07 0.57 0.52 0.56
Moser 0.08 0.56 0.50 0.57
Contois 0.00 0.02 0.35 0.56
Firstorder 0.07 0.57 0.52 0.56
Secondorder 0.85 0.80 0.98 0.77
Thirdorder 0.88 0.85 0.95 0.68
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decreased into 0.5 g per kg soil concentration [23]. more amount of mazut was degraded. Addition of every
Using gradual addition of mazut method caused quick concentration of mazut could increase degradation but
growth and adaptation of bacteria at initial days. Thus onlyuptocertainamountinwhichincreaseinconcentra-
biodegradation of mazut carried out by most possible tion would not raise degradation capacity. After 21 days
amount of bacteria nevertheless in simple method degradationofheavycrudeoilbyE.cloacaeat50000ppm,
amount of bacteria became maximal at the end of 10000 ppm and 2500 ppm concentrations were %10, %40
process, and due to that gradual addition increased bio- and %53 respectively. At high concentrations of heavy
degradation of mazut to %10 more. Increase of mazut crude oil degradation percentage did not change and in-
concentration resulted increase in efficiency of degra- creasing concentration decreased microbial growth and
ded mazut; nevertheless percentage of degradation degradation percentage [11]. Usually in biodegradation of
decreased. The changes due to increasing concentration hydrocarbons, increase of hydrocarbon concentration
were not linear and whatever percentage of increase of raised degraded hydrocarbon and reduced percentage of
mazut was more, percentage of degradation fell down degradation[24,28-31];neverthelessincreaseofconcentra-
progressively since at very high concentrations increase tionwasabletodecrease[24,25]orincrease[31]microbial
ofconcentration hadnoeffectsondegradation. growth.
Changes in pH of medium were appropriate for bac- In general, comparing with other studies we could able
terial growth and activity. Simultaneously with higher more clearly show the effective mazut degradation by
rate of microbial growth, reduction in value of pH was gradualadditionalmethodbecausethereisnotanyreport
faster and medium became more acidified. This published about kinetics of biodegradation of mazut. This
phenomenon acted as an inhibitor factor for bacterial study could be first in this type of investigation and more
growth and caused stopping growth. After fastgrowth of studiescanbecarriedoutinfuture.Thebiokineticmodels
bacteria at starting days, no growth was observed when could be most powerful models, although were not so
pH unchanged and became constant. In this study, most much correlated with biodegradation of mazut; hence,
amount of reduction of pH was about 1 unit in all of the there is demand for new more powerful model to predict
assays. During the biodegradation of mazut, materials in mazutbiodegradationprocess.
medium were chemically changed as conductivity of
medium was affected by it. Biodegradation of mazut Conclusion
resulted increase of medium’s electric potential that was Mazutwereefficientlydegradedbynewlyisolatedbacteria.
maximum at growth time. Usually in degradation of Mazut biodegradation was improved with gradually
hydrocarbons, increase of microbial activities can cause addition of substrate. Substrate addition allowed bacteria
quick reduction of pH which is a reason for reduction of to gradually adapt to a heavy hydrocarbon medium. Gra-
microbial growth; as at following process pH was con- dually adaptation raised biodegradation rate, increasing
stant [24-27]. Electric potential is a parameter which biomass.Increasingmazut concentration reduced degrad-
assignsamount of ions. Changinginions amount directly ation percentage because bacterial growth and conse-
relates to change in electric potential in each medium. quently, degrading enzymes production were limited in a
Microbialactivitiesalternateionsamountbutchangesare batch medium. Changing in pH and electric potential of
different in relation to each ion in a medium. Basically, medium were fluctuated during biodegradation. In the
ionic changes in bacterial activities are affected with earlyday,decreaseofpHandincreaseofelectricpotential
hydrogen ions, because changes in other ions, which are wereoccurred;then, their oscillationswerereduced. Elec-
consumed as nutrients or are produced, are not sensible. tricpotentialchangecouldbeevidenceforpHalternation,
Thus, decrease of pH, increasing hydrogen ions, practic- which can help to tune pH in the medium without mea-
ally raises electric potential. In this study, pH changes di- suringpH.Investigationofmathematicalmodelsonmazut
rectly affected on electric potential. Whatever rate of pH biodegradationstated complexityofmazutbiodegradation
changes became higher, rate of electric potential changes to mathematically modeling. Therefore, modeling mazut
got higher. The recorded data demonstrated close rele- degradation, and using substrate addition technique can
vance between pH and electric potential changes. Conse- bestudiedtoimproveotherbiodegradationprocesses.
quently, measuring electric potential in medium can help
estimating pH without measuring it. On the other hand, Competinginterests
Theauthorsdeclarethattheyhavenocompetinginterests.
comparing pH trend with electric potential trend can
indicate sensible presence of other ionic materials during Authors’contributions
microbialactivities. ACK,carriedoutthisstudyaspartofhisM.Sc.project,planedtheoriginal
Increaseofmazutconcentrationinducedbacteriatocon- researchoftheproject,andwrotethemanuscript.MM,supervisedthe
microbialpartofthisresearch,andrevisedthemanuscript.SY,supervised
sume more mazut. Results showed that addition of mazut
themathematicalpartoftheproject,andreadthemanuscript.Allauthors
into medium decreased percentage of biodegradation but readandapprovedthefinalmanuscript.
Khorasanietal.IranianJournalofEnvironmentalHealthSciences&Engineering2013,10:2 Page7of7
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