role of hydrocarbon degrading bacteria serratia marcescens ace2 and bacillus cereus ace4 on...

Published: July 30, 2011

r 2011 American Chemical Society 10041 dx.doi.org/10.1021/ie200709q | Ind. Eng. Chem. Res. 2011, 50, 10041–10046

ARTICLE

pubs.acs.org/IECR

Role of Hydrocarbon Degrading Bacteria Serratia marcescens ACE2and Bacillus cereus ACE4 on Corrosion of Carbon Steel API 5LXAruliah Rajasekar,*,† Rajasekhar Balasubramanian,† and Joshua VM Kuma‡

†Department of Civil and Environmental Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576‡Minerals, Metals, andMaterials TechnologyCentre (M3TC), National University of Singapore, Faculty of Engineering, Singapore 117576

ABSTRACT:This paper reports themicrobiologically induced corrosion (MIC) and electrochemical behavior of carbon steel (API5LX) in the presence of hydrocarbon-degrading bacteria Bacillus cereus ACE4 (a Gram-positive bacterium) and Serratia marcescensACE2 (a Gram-negative bacterium). Weight loss studies and metallographic analysis of the metal API 5LX exposed to a simulatedcorrosive environment showed that the bacterium ACE4 caused severe pitting corrosion than that of bacterium ACE2. As part ofbiodegradation studies, the impact of aryl hydrocarbon hydroxylase (AHH) on diesel degradation was investigated along withreduction of total hydrocarbons. It was clearly observed that, during the biodegradation experiment in the presence of B. cereusACE4, the content of the total hydrocarbons decreased significantly due to their metabolism induced by AHH enzymes whencompared to S. marcescens ACE2. Degraded petroleum hydrocarbons (diesel) act as a good nutrient for bacteria, which in turnincreases the proliferation of bacteria on the steel and determines the nature of corrosion. Metal oxides such as MnO2 and Fe2O3

were found as part of the corrosion products, indicating that the ACE4 bacterium is capable of converting the elements on the carbonsteel (API 5LX) to their metal oxides and thus accelerating severe pitting corrosion on the surface of the pipeline networks. Overall,the study provides an insight into themicrobiologically influenced corrosion of carbon steel API 5LX by two hydrocarbon-degradingbacteria in diesel fuel/water mixtures.

1. INTRODUCTION

Microbiologically induced corrosion (MIC) is one of the welldocumented phenomena in corrosion, which causes a deleteriouseffect on petroleum product pipeline, storage tanks, and variousindustries.1�3 MIC affects the operation and maintenance costsof the pipelines, and many oil pipelines experience severe corrosionandmicrofouling problems.1 It has been estimated that 40% of allinternal pipeline corrosion in the petroleum industry can beattributed to MIC.2 Carbon steel is a commonly used engineer-ing material of construction, and leakage of petroleum hydro-carbons such as diesel due to the internal corrosion of carbonsteel tanks has been well documented in many countries aroundthe world such as USA, France, Sweden, Switzerland, and India.4�6

The diverse groups of bacteria have been associated withhydrocarbon degradation.7 Hence, the role of bacteria in thedegradation of petroleum hydrocarbons caused byMIC needs tobe thoroughly investigated in order to protect petroleum productpipelines. However, there are only a few reports available in theliterature addressing the involvement of individual bacterialspecies in diesel degradation as induced by MIC.

This is thefirst study that has investigated the role of hydrocarbondegrading bacteria, Bacillus cereus ACE4 and Serratia marcescensACE2 bacterial enzymes, in MIC and its impact on the biode-gradation of diesel as relevant to a tropical country pipeline; ACE2and ACE4 refer to the respective strain number of individualbacterial species isolated from the petroleum products trans-ported via the pipeline. Previous microbiological studies haveconcluded that sulfate-reducing bacteria (SRB) play a major rolein MIC.5,8�12 Phylogenetic characterization and environmentalscanning microscopy (SEM) analysis of corrosive consortium ofbacteria revealed a low abundance of SRB in sour gas pipelines.13

These studies demonstrated that SRB need not be present inabundance in all microbial communities responsible for MIC inthe petroleum industry. The low abundance of SRB can partly beexplained by the relatively high flow velocity in pipelines whichmay create uniform distribution of oxygen, leading to the suppres-sion of the growth of SRB.13,14 Under the low abundance of SRB,the ability of enteric bacterium, Serratia marcescens ACE2, todegrade petroleum hydrocarbon appears to be an alternativeexplanation since this feature has always been associated withtypical soil containing such bacteria.15 Muthukumar et al.7 reportedthat Brucella sp. and Gallionella sp. could degrade diesel in atransporting pipeline in Northwest India, while Rajasekar et al.16

detected bacterial genera Pseudomonas sp, Bacillus sp, Gallionellasp, Siderocapsa sp,Thiobacillus sp,Thiospira sp, Sulfolobus sp., andVibrio sp. in a naphtha pipeline. The latter group also reportedinteractions between heterotrophs and chemolithotrophs innaphtha-transporting pipelines in Southwest India.

The study addressed in this article provides new insights intodegradation of diesel fuels by enteric bacteria and demonstratesthe need for a comprehensive understanding of metabolic andphysiological properties of enteric bacterium (ACE2) duringpetroleum hydrocarbon degradation. The outcome of the studycan help in developing and using efficient and effective biore-mediation strategies. However, only a handful of reports addressdegradation of aromatic compounds by Enterobacteria, particu-larly those of the genera Klebsiella, Escherichia, and Hafnia.15

Received: April 6, 2011Accepted: July 30, 2011Revised: June 28, 2011

10042 dx.doi.org/10.1021/ie200709q |Ind. Eng. Chem. Res. 2011, 50, 10041–10046

Industrial & Engineering Chemistry Research ARTICLE

Although there are several reports on bioremediation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs),research pertaining to biodegradation of these substances by entericbacteria has been relatively sparse.15 Hunter et al.17 made anattempt to find out whether soil bacteria Bacillus sp. are capableof degrading high molecular weight PAHs, such as pyrene (Pyr)and benzo[a]pyrene (BaP). It was found that Bacillus subtilis wasable to transform approximately 40% and 50% pyrene andbenzo[a]pyrene, respectively. The report by Hunter et al. repre-sents the first report implicating B. subtilis in PAH degradation.Hence, the present study attempted to study the specific role ofaromatic hydrocarbon hydroxylase (AHH) enzymes present amongour bacterial strains in the degradation process while incubatingwith diesel.

The objective of the current study was to investigate the effectof bacterial contaminants on the biodegradation and corrosionbehavior of carbon steel, using a Gram-positive bacterium, Bacilluscereus ACE4, and a Gram-negative bacterium, Serratia marcescensACE2. The potential of these two hydrocarbon-degrading bacteriato corrode carbon steel was determined using weight loss studiesand surface analysis (SEM and X-ray diffraction (XRD)) techni-ques. The impact of aryl hydrocarbon hydroxylase (AHH) on dieseldegradation has been investigated by analyzing the reduction of totalhydrocarbons using a fluorescent spectrophotometer.

2. MATERIALS AND METHODS

2.1. Microorganisms. S. marcescens ACE2 and Bacillus cereusACE4 were isolated from a corrosion product at the diesel-transporting pipelines in a northwestern region of India andidentified as described earlier.18 The 16S rDNA gene analysis andthe nucleotide sequence data of ACE2 and ACE4 were depositedin GenBank under accession numbers DQ092416 and AY912105.The ability of these organisms to grow on hexadecane was deter-mined by inoculating bacterial isolates into test tubes with sterileBushnell-Hass medium (BH). BH, containing inorganic nutrientssuch as magnesium sulfate, 0.20 g/L; calcium chloride, 0.02 g/L;monopotassium phosphate, 1 g/L; dipotassium phosphate, 1 g/L;ammonium nitrate, 1 g/L; ferric chloride, 0.05 g/L; chloride,120 ppm; pH 7, was supplemented with 1% diesel as the solecarbon source to enumerate total heterotrophs and hydrocarbon-degrading microorganisms. Diesel samples were sterilized byfiltering through a Millipore 0.45 μm pore size membrane filter.Cultures were shaken at 100 rpm at 25 �C, and growth wasdetermined by measuring optical density at 400 nm (UV�visspectrophotometer, Shimadzu BioSpec mini). The culture wasrecharacterized on the basis of the following analyses: morphol-ogy,Gram staining, spore staining,motility, oxidase, catalase, oxidativefermentation, gas production, ammonia formation, nitrate andnitrite reduction, indole production test, methyl red and Voges-Proskauer tests, citrate andmannitol utilization test, hydrolysis ofcasein, gelatin, starch, urea, and lipid.19

2.2. Biodegradation Study and Aryl Hydrocarbon Hydro-xylase Assay. Bacterial cultures ACE4 and ACE2 preculturedovernight at 30 �C in BH broth medium were transferred to a250mL Erlenmeyer screw capped flask (to prevent loss of volatilediesel hydrocarbon) containing 100 mL of BH and 10 g L�1 diesel.Cells of ACE2 and ACE4 were incubated aerobically at 30 �C ona rotary shaker operated at 150 rpm for 30 days, and dieselhydrocarbons remaining in the culture medium were deter-mined. The control (uninoculated) was incubated parallel withthe experimental system to monitor abiotic losses of the diesel

substrate. All the experiments which included uninoculatedcontrols were performed in duplicate. After the biodegradationexperiment in the presence/absence of bacteria ACE2 and ACE4,the diesel was extracted with hexane solvent for total petroleumhydrocarbon analysis (TPH). The total hydrocarbon contents inthe diesel concentrations were determined with a fluorescencespectrophotometer at excitation wavelength 270 nm and emis-sion at 330 nm by the standard method.20 Aryl hydrocarbonhydroxylase (AHH) wasmeasured in bacterial samples as describedby Dehnen et al.21 All assays were carried out in the presence ofNADPH (reduced β-nicotinamide adenine dinucleotide phos-phate) and measured fluorometrically (excitation 460 nm, emis-sion 517 nm) under yellow light. One milliliter of assay volumecontains 100 mM triethanolamine�HCl (pH 7.25), 4 mMNADPH, 60 μM benzo[a]pyrene, and 100 μL of microsomalhomogenate, which was incubated for 10 min. Arbitrary fluores-cence units were converted into picomoles of benzo[a]pyrene-phenols formed using intercalibration between quinine sulfateand 3-hydroxybenzo[a]pyrene. Themetabolite 3-hydroxybenzo-[a]pyrene was obtained from the NCI Chemical CarcinogenRepository, USA.2.3. Corrosion Studies and Surface Analysis. Carbon steel

API 5LX (C-0.29 max, S-0.05 max, P-0.04 max, Mn-1.25 max.)coupons (2.5 cm � 2.5 cm) were sequentially ground with aseries of grit silicon carbide papers (grades 180, 500, 800, 1200,and 1500) to a smooth surface and were finally polished to amirror finish surface using 0.3 μm alumina powder.8 The polishedcoupons were rinsed with deionized water and then degreasedwith trichloroethylene. In the present study, system 1 consistedof 500 mL of diesel with 2% water containing 120 ppm chlorideand 1% BH broth as control. Systems 2 and 3 consisted of system1 inoculated with 2 mL of ACE2 and ACE4 as experimentalsystems, respectively, at about initial load of 106 CFU/mL foreach system. The colony forming units (CFU) per milliliter weredetermined using the standard serial dilution method followedby the pour plate technique. Biocorrosion experiments wereinitiated by hanging pristine coupons on a nylon string in boththe medium with and without the bacteria. After the incubationof the fifth and tenth days, the coupons were removed andpickled in Clark solution (2% antimony trioxide + 5% stannouschloride dissolved in concentrated HCl at room temperaturewith constant stirring about 5�10 min) and washed in water anddried with an air drier. Duplicate experiments were made for eachsystem. Final weights of the six coupons in each system weretaken, and the average corrosion rates were calculated as recom-mended by the National Association of Corrosion Engineers(NACE), Houston. The standard deviation for each system ispresented. The pHwasmeasured at different time periods (5 and10 days) after the weight loss method in the presence of bacteria.A computer controlled powder X-ray diffractometer (XRD,X0per PRO (PANalytical model)) was used to scan the corrosionproducts between 100 and 850 � 2θ Cu K (2.2 KW maximum)and with R radiation (Ni filter) at a rating of 40 KV, 20 mA. Thedried corrosion products were collected at the end of theincubation period, i.e., 10th day, crushed into a fine powder,and used for XRD analysis for determining the nature ofoxides present in the corrosion product. The surface morpho-logical characteristics of the control and experimental cou-pons API 5LX were observed under a scanning electronmicroscope (SEM) (Hitachi model S-3000H) at a magnifica-tion ranging from 50� to 200� operated at an acceleratingvoltage of 25 kV.22



2.4. Electrochemical Analysis. For electrochemical studies, amixture of diesel oil and water (containing 120 ppm chloride ion)in the ratio 2:1 was made.23 The API 5LX steel coupon wasembedded in Araldite with an exposed area of 1.0 cm2 as aworking electrode. In the present study, 75 mL of 1% BH broth(containing 120 ppm chloride) and 150 mL of diesel oil wereused as the control system, while 75 mL of 1% BH broth(containing 120 ppm chloride) and 150 mL of diesel 500 mLdiesel inoculated with 2 mL of inoculum ACE2 and ACE4 about104 CFU/mL were used as the experimental systems 2 and 3.The mixtures were stirred vigorously for about 120 h. After the10th day, the coupons were removed from the respective systemsand potentiodynamic polarization was carried out using poten-tiostate model PGP201 with volta master-1-software. A couponofAPI 5LX1 cm2 asworking electrode, a standard calomel electrode(SCE) as reference electrode, and a platinum wire as counterelectrode were employed for the polarization study. Tafel curveswere measured with a scan rate of 0.5 mV s�1 and were obtainedby scanning from the open circuit potential (Ecorr) toward 200 mVanodically and �200 mV cathodically using duplicate coupons.

3. RESULTS AND DISCUSSION

The total hydrocarbon at the initial stage was about 30mg/mLin the absence of bacteria. After degradation by ACE2, the totalhydrocarbon content was 10 mg/mL while in the presence ofACE4 the total hydrocarbon was 8.7 mg/mL. This may be due tothe presence of higher activity of AHH in ACE4, and it furtherindicates that the activity of ACE4 on degradation of diesel wasmore severe than that of ACE2. The activities of AHH weremeasured during biodegradation of diesel. In the stage ofbiodegradation, the activity of AHH in ACE2 was measured as11.38 p mol/min. After 20 days of bacterial inoculation in diesel,the quantity of AHH decreased significantly to below thedetection limit (BDL, <0.001 pmol/min). Similarly, in the caseof ACE4, the activity of AHH was 15.91 p mol/min, whichdecreased to BDL after 20 days. This observation indicates that atthe initial stage of biodegradation the inoculated bacteria mayproduce a large quantity or detectable levels of AHH enzyme.24,25

The Bacillus species-mediated conversion of PAHwith 3�5 rings(acenaphthene, fluoranthene, pyrene, benzo[e]pyrene) was de-monstrated earlier.26 Aitken et al.27 also observed mineralizationof benzo(a)pyrene (one of the PAHs) by different types ofbacterial strains. Since diesel is the major organic nutrient sourcefor bacteria, they consume the carbon from diesel with the help ofhydrocarbon metabolizing enzymes such as AHH. After degra-dation, the availability of simple molecules in diesel or metabo-lites of diesel/hydrocarbons may inhibit AHH activity and act assubstrate inhibitor.28,29

In addition to the enzyme assay discussed above, we measuredthe content of total hydrocarbons in diesel at the beginning stageand after 20 days of incubation with ACE2 and ACE4. It has beenclearly observed that during the biodegradation experiment thecontent of the total hydrocarbon is totally decreased or metabo-lized by AHH enzymes at the 20th day. It can be concluded thatACE4 isolate efficiently degraded aromatic hydrocarbon chainsin diesel oil. It has been reported that, in general, Gram-positivebacteria (ACE4) secrete more lipoteichoic acids (LTAs) comparedto Gram-negative bacteria (ACE2).30 The abundant availability ofLTAs released from ACE4 been implicated in hydrophophobicinteractions.30,31 It has been shown that LTAs are releasedintracellularly from their initial site on the surface of the cyctoplasmicmembrane. Following their release, LTAs may adopt a chemicalconformation in which ionic interactions between their poly-glycerolphosphate chain and cell wall components alter theirorientation in such a way that their lipid portion is exposedtoward the outer surface, making ACE4 hydrophobic and thusinducing the degradation of hydrocarbons in diesel.30,31

Weight loss data obtained in the presence/absence of bacteriaACE2 and ACE4 are presented in Table 1. In the control system(uninocualted), the weight loss was 3.82 mg after 5 days and11.12 mg after 10 days whereas in the presence of Bacillus cereusACE4 the weight loss was 6.96 mg on the fifth day and 23.55 mgon the tenth day. In the presence of S. marcescens, ACE2 weightloss was 11.3 mg on the fifth day and was 21.13 mg on the tenthday. However, in the presence of ACE4, the corrosion rate washigher than that with ACE2 as shown in Table 1. The pH of theACE2 inoculated system was 5.89 and that of ACE4 was 6.32.The lowering of pH can be attributed to the release of organicacids from the corresponding bacterial strains furing the course ofbiodegradation of diesel.

The data obtained from the polarization study in the pre-sence/absence of bacteria ACE2 and ACE4 are presented inTable 2. The open circuit potential (OCP) of API 5LX in controlsystem was �660 mV vs SCE (saturated calomel electrode)while, in the presence of both bacterial systems, the potentialshifted to the negative side to about�711 mV vs SCE and�720mV vs SCE. The corrosion current for API 5LX in the controlsystem was about 8.69 � 10�6 A/cm2 whereas, in bacterialsystems ACE2 and ACE4, the corrosion current increased toabout 3.76� 10�5 A/cm2 and 4.50� 10�5 A/cm2, respectively.There was no significant difference in the cathodic Tafel slope(bc) value between control and experimental systems. However,the anodic Tafel slope (ba) value for the control system was186 mV/decade and in the experimental system was 157 mV/decade for ACE2 and 167 mV/decade for ACE4. This observa-tion indicates that the anodic current increased between thepotential �660, �711, and �720 mV vs SCE by both bacteria.

Table 1. Corrosion Rate of Carbon Steel API 5LX in the Various Corrosive Systems

s.

no. systems

incubation

periods

(days)

weight

loss

(mg)

corrosion

rate

(mm/year)

1 500 mL diesel + 2% water containing 120 ppm chloride and 1% BH broth 5 3.82 ( 2.18 0.027

10 11.12 ( 1.21 0.039

2 500 mL diesel + 2% water containing120 ppm chloride and 1% BH broth + 2 mL ACE2 inoculum about 106 CFU/mL 5 6.96 ( 1.19 0.050

10 23.55 ( 2.28 0.084

3 500 mL diesel + 2% water containing120 ppm chloride and 1% BH broth + 2 mL ACE4 inoculum about 106 CFU/mL 5 11.31 ( 2.25 0.081

10 21.13 ( 1.12 0.075



These observations suggest that bacteria induce the corrosion ofsteel by oxidation of degraded organic and iron species in the metalsurfaces which supports the observationmade by Rajasekar et al.16

Figure 1 presents the details of XRD data corresponding tothe phases present in the corrosion product sample collectedfrom control and experimental systems. R-Iron oxide hydroxide(R-FeO(OH), Fe(OH)2) was observed in the control system(Figure 1a). Peaks with high intensity were observed correspond-ing to R-ferric oxide (Fe2O3), R-iron oxide hydroxide R-FeO-(OH), andλ-manganesedioxide (λ-MnO2) in theACE2(Figure 1b)

and ACE4 inoculated systems (Figure 1c). XRD results reveal thepresence of ferric oxides (Fe2O3) andmanganese oxides (λ-MnO2)indicating the role of ACE2 and ACE4 on manganese/iron

Table 2. Polarization Data for Carbon Steel API 5LX in the Presence/Absence of Bacteria

systems Ecorr (mV vs SCE) ba (mV/decade) bc (mV/decade) Icorr (A/cm2)

control system 1 �660 186 248 8.69 � 10�6

S. marcescens ACE2 inoculated system 2 �711 157 238 3.76 � 10�5

B. cereus ACE4 inoculated system 3 �720 167 332 4.50 � 10�5

Figure 1. XRD pattern of corrosion products collected from varioussystems: (a) uninoulated system; (b) experimental system, inoculatedwith S. marcescens ACE2; (c) experimental system, inoculated withB. cereus ACE4.

Figure 2. SEM images of carbon steel API 5 LX. (a) Metal exposed tothe medium without bacteria; (b) metal exposed to the medium withS. marcescens ACE2; (C) metal exposed to the medium with ACE4.



deposition during the formation of corrosion products andin the acceleration of the microbial corrosion directly on thepipeline. These bacteria are capable of oxidizing manganous(Mn2+) ions, ferrous (Fe2+) ions into manganic (Mn4+) ions andferric (Fe3+) ions with concomitant deposition of MnO2, Fe2O3

followed by dense accumulation on the metal surface, leading tocorrosion. Jones et al.32 suggest that the insoluble product is amixture of ferric oxide (Fe2O3) and ferric hydroxide (Fe(OH)3),which can be referred to as FeO(OH)n. Jones et al.

32 explainedthat the ferric ions are unlikely to precipitate completely, especiallyin the acidic crevice regions (pH was in the range of 5.0 to 6.0 inboth bacterial systems when compared to the control systemwith pH 6.8). As a consequence, ferric ions remaining in solutionserve as highly oxidizing species and thus tend to acceleratecorrosion. Moreover, in waters containing chloride ions, iron-oxidizing bacteria may be directly involved in the production offerric chloride (FeCl3) which is an extremely corrosive substancethat can concentrate under nodules.33,34 Chloride ions can migrateinto a crevice location by neutralizing the increased charge viaanodic dissolution and then combining with the oxidized prod-ucts of ferrous and manganous ions, as evident from Figure 1c.

The surface morphological characteristics of the control andexperimental steel API 5LX were inoculated with ACE2 andACE4; API 5LX coupons were studied under a scanning electronmicroscope (SEM). Figure 2 shows the SEM images of the API5LX steel coupons after 10 days of exposure in the bacteria inoculatedsystemACE2 and ACE4 and uninoculated system after removingof corrosion products and biofilm. Uniform corrosion (moderatepit formation) was observed in the control system (Figure 2a)whereas in the experimental system (ACE2 and ACE4 inoculatedsystem) severe pitting attacks showing a pit size of more than2μm in diameter were observed over the surface of steel (Figure 2b,c).It is thus clear that the bacteria ACE2 and ACE4 induce thecorrosion process by formation of Fe2O3. In addition, the degradedcarbon (diesel) acts as a good nutrient for bacteria, which increasesthe proliferation of bacteria on the steel surface and determinesthe nature and degree of degradation and corrosion35 as shownschematically below.

O2ðsolÞ þ 2H2O þ 2e� f 2H2O2ðadsÞ ð1Þ

2H2O2ðsolÞ f H2O þ 1=2O2ðsolÞ ð2Þ

Overall, the mechanism involved in the biocorrosion of car-bon steel API 5LX can be described as in the above equations.B. cereus ACE4 and S. marcescens ACE2 are facultative anaerobes,

and biochemical tests indicate the presence of catalase andcytochrome oxidase.18 Both ACE2 and ACE4 have a peroxidaseenzyme, which produces hydrogen peroxide that is involved inthe degradation of hydrocarbon fuel. The bacteria also produce acatalase, which overcomes the toxic nature of hydrogen peroxide(reaction 1) by breaking it down intowater andoxygen (reaction 2).During respiration by these isolates, oxygen is consumed andhydrogen is utilized from the degraded hydrocarbon product.36

The oxygen radical produced during the bacterial metabolismbinds to the iron metal atom present on the surface of the carbonsteel and forms a superoxide surface anion radical (reaction 3).The species in solution (sol) or adsorbed to the surface (ads) areindicated in the following chemical reactions. Finally, the surfaceanion interacts with water, which leads to the oxidation of ferricto ferric oxides as corrosion products, along with the hydroxideanion (reaction 4). The oxidative properties of H2O2 by thehydrocarbon-degrading bacteria led to the conversion of corrosionproducts as iron oxides in carbon alloy.37,38 The overall resultsobtained support the hypothesis that the microbially inducedcorrosion of carbon steel occurs through the participation of Fe2O3

which is a result of the degradation of hydrocarbons in diesel.

4. CONCLUSIONS

The influence of hydrocarbon-degrading bacterial biofilmS. marcescens ACE2 and B. cereusACE4 strains on API 5LX carbonsteel exposed to diesel/water mixtures in 120 ppmNaCl solutionhas been examined in detail using the weight loss method, surfaceanalysis (SEM and XRD). In addition, the biodegradationstudies, the impact of aryl hydrocarbon hydroxylase enzyme onaromatic hydrocarbon degradation has been estimated usingfluorescence measurement. B. cereus ACE4 isolate efficientlydegraded aromatic hydrocarbon chains in diesel oil when com-pared to ACE2 due to the more secretion of aryl hydrocarbonhydrooxlase enzyme. It can therefore be concluded that Gram-positive bacteria (ACE4) secrete more lipoteichoic acids (LTAs)than Gram-negative bacteria (ACE2). LTAs have been impli-cated in promoting hydrophophobic interactions, leading tofaster degradation of petroleum hydrocarbons. In the presenceof ACE4, the corrosion current density, icorr (4.50 � 10�6 A/cm2), was much higher than in the control (8.69 � 10�10 A/cm2) and ACE2 (3.76� 10�10 A/cm2) systems. The presence ofMnO2 and Fe2O3 as part of the corrosion products indicated thatthe Gram-positive bacteria are capable of converting the ele-ments, manganese and iron, on the carbon steel (API 5LX) totheir metal oxides and accelerating severe pitting corrosion onthe surface of the pipeline networks. Even though ACE2 andACE4 bacteria may be useful in the bioremediation of a diesel-polluted habitat, their presence in diesel pipeline and transporta-tion facilities would lead to the reduction in the quality of dieseland in turn severe economic loss.

’AUTHOR INFORMATION

Corresponding Author*Tel.: +65 65165135. E-mail: [email protected] or [email protected].

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role of hydrocarbon degrading bacteria serratia marcescens ace2 and bacillus cereus ace4 on...

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