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Role of Hydrocarbon Degrading Bacteria Serratia marcescens ACE2 and Bacillus cereus ACE4 on Corrosion of Carbon Steel API 5LX Aruliah 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, and Materials Technology Centre (M3TC), National University of Singapore, Faculty of Engineering, Singapore 117576 ABSTRACT: This paper reports the microbiologically induced corrosion (MIC) and electrochemical behavior of carbon steel (API 5LX) in the presence of hydrocarbon-degrading bacteria Bacillus cereus ACE4 (a Gram-positive bacterium) and Serratia marcescens ACE2 (a Gram-negative bacterium). Weight loss studies and metallographic analysis of the metal API 5LX exposed to a simulated corrosive environment showed that the bacterium ACE4 caused severe pitting corrosion than that of bacterium ACE2. As part of biodegradation studies, the impact of aryl hydrocarbon hydroxylase (AHH) on diesel degradation was investigated along with reduction of total hydrocarbons. It was clearly observed that, during the biodegradation experiment in the presence of B. cereus ACE4, the content of the total hydrocarbons decreased significantly due to their metabolism induced by AHH enzymes when compared to S. marcescens ACE2. Degraded petroleum hydrocarbons (diesel) act as a good nutrient for bacteria, which in turn increases 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 carbon steel (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 the microbiologically influenced corrosion of carbon steel API 5LX by two hydrocarbon-degrading bacteria in diesel fuel/water mixtures.
1. INTRODUCTION Microbiologically induced corrosion (MIC) is one of the well documented phenomena in corrosion, which causes a deleterious effect on petroleum product pipeline, storage tanks, and various industries.13 MIC affects the operation and maintenance costs of the pipelines, and many oil pipelines experience severe corrosion and microfouling problems.1 It has been estimated that 40% of all internal pipeline corrosion in the petroleum industry can be attributed to MIC.2 Carbon steel is a commonly used engineering material of construction, and leakage of petroleum hydrocarbons such as diesel due to the internal corrosion of carbon steel tanks has been well documented in many countries around the world such as USA, France, Sweden, Switzerland, and India.46 The diverse groups of bacteria have been associated with hydrocarbon degradation.7 Hence, the role of bacteria in the degradation of petroleum hydrocarbons caused by MIC needs to be thoroughly investigated in order to protect petroleum product pipelines. However, there are only a few reports available in the literature addressing the involvement of individual bacterial species in diesel degradation as induced by MIC. This is the first study that has investigated the role of hydrocarbon degrading bacteria, Bacillus cereus ACE4 and Serratia marcescens ACE2 bacterial enzymes, in MIC and its impact on the biodegradation of diesel as relevant to a tropical country pipeline; ACE2 and ACE4 refer to the respective strain number of individual bacterial species isolated from the petroleum products transported via the pipeline. Previous microbiological studies have concluded that sulfate-reducing bacteria (SRB) play a major role in MIC.5,812 Phylogenetic characterization and environmental scanning microscopy (SEM) analysis of corrosive consortium of bacteria revealed a low abundance of SRB in sour gas pipelines.13 r 2011 American Chemical Society
These studies demonstrated that SRB need not be present in abundance in all microbial communities responsible for MIC in the petroleum industry. The low abundance of SRB can partly be explained by the relatively high flow velocity in pipelines which may create uniform distribution of oxygen, leading to the suppression of the growth of SRB.13,14 Under the low abundance of SRB, the ability of enteric bacterium, Serratia marcescens ACE2, to degrade petroleum hydrocarbon appears to be an alternative explanation since this feature has always been associated with typical soil containing such bacteria.15 Muthukumar et al.7 reported that Brucella sp. and Gallionella sp. could degrade diesel in a transporting pipeline in Northwest India, while Rajasekar et al.16 detected bacterial genera Pseudomonas sp, Bacillus sp, Gallionella sp, Siderocapsa sp, Thiobacillus sp, Thiospira sp, Sulfolobus sp., and Vibrio sp. in a naphtha pipeline. The latter group also reported interactions between heterotrophs and chemolithotrophs in naphtha-transporting pipelines in Southwest India. The study addressed in this article provides new insights into degradation of diesel fuels by enteric bacteria and demonstrates the need for a comprehensive understanding of metabolic and physiological properties of enteric bacterium (ACE2) during petroleum hydrocarbon degradation. The outcome of the study can help in developing and using efficient and effective bioremediation strategies. However, only a handful of reports address degradation of aromatic compounds by Enterobacteria, particularly those of the genera Klebsiella, Escherichia, and Hafnia.15 Received: April 6, 2011 Accepted: July 30, 2011 Revised: June 28, 2011 Published: July 30, 2011 10041
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Industrial & Engineering Chemistry Research Although there are several reports on bioremediation of highmolecular-weight polycyclic aromatic hydrocarbons (PAHs), research pertaining to biodegradation of these substances by enteric bacteria has been relatively sparse.15 Hunter et al.17 made an attempt to find out whether soil bacteria Bacillus sp. are capable of degrading high molecular weight PAHs, such as pyrene (Pyr) and benzo[a]pyrene (BaP). It was found that Bacillus subtilis was able to transform approximately 40% and 50% pyrene and benzo[a]pyrene, respectively. The report by Hunter et al. represents the first report implicating B. subtilis in PAH degradation. Hence, the present study attempted to study the specific role of aromatic hydrocarbon hydroxylase (AHH) enzymes present among our bacterial strains in the degradation process while incubating with diesel. The objective of the current study was to investigate the effect of bacterial contaminants on the biodegradation and corrosion behavior of carbon steel, using a Gram-positive bacterium, Bacillus cereus ACE4, and a Gram-negative bacterium, Serratia marcescens ACE2. The potential of these two hydrocarbon-degrading bacteria to corrode carbon steel was determined using weight loss studies and surface analysis (SEM and X-ray diffraction (XRD)) techniques. The impact of aryl hydrocarbon hydroxylase (AHH) on diesel degradation has been investigated by analyzing the reduction of total hydrocarbons using a fluorescent spectrophotometer.
2. MATERIALS AND METHODS 2.1. Microorganisms. S. marcescens ACE2 and Bacillus cereus ACE4 were isolated from a corrosion product at the dieseltransporting pipelines in a northwestern region of India and identified as described earlier.18 The 16S rDNA gene analysis and the nucleotide sequence data of ACE2 and ACE4 were deposited in GenBank under accession numbers DQ092416 and AY912105. The ability of these organisms to grow on hexadecane was determined by inoculating bacterial isolates into test tubes with sterile Bushnell-Hass medium (BH). BH, containing inorganic nutrients such 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 sole carbon source to enumerate total heterotrophs and hydrocarbondegrading microorganisms. Diesel samples were sterilized by filtering through a Millipore 0.45 μm pore size membrane filter. Cultures were shaken at 100 rpm at 25 °C, and growth was determined by measuring optical density at 400 nm (UVvis spectrophotometer, Shimadzu BioSpec mini). The culture was recharacterized on the basis of the following analyses: morphology, Gram staining, spore staining, motility, oxidase, catalase, oxidative fermentation, gas production, ammonia formation, nitrate and nitrite reduction, indole production test, methyl red and VogesProskauer tests, citrate and mannitol utilization test, hydrolysis of casein, gelatin, starch, urea, and lipid.19 2.2. Biodegradation Study and Aryl Hydrocarbon Hydroxylase Assay. Bacterial cultures ACE4 and ACE2 precultured overnight at 30 °C in BH broth medium were transferred to a 250 mL Erlenmeyer screw capped flask (to prevent loss of volatile diesel hydrocarbon) containing 100 mL of BH and 10 g L1 diesel. Cells of ACE2 and ACE4 were incubated aerobically at 30 °C on a rotary shaker operated at 150 rpm for 30 days, and diesel hydrocarbons remaining in the culture medium were determined. The control (uninoculated) was incubated parallel with the experimental system to monitor abiotic losses of the diesel
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substrate. All the experiments which included uninoculated controls were performed in duplicate. After the biodegradation experiment in the presence/absence of bacteria ACE2 and ACE4, the diesel was extracted with hexane solvent for total petroleum hydrocarbon analysis (TPH). The total hydrocarbon contents in the diesel concentrations were determined with a fluorescence spectrophotometer at excitation wavelength 270 nm and emission at 330 nm by the standard method.20 Aryl hydrocarbon hydroxylase (AHH) was measured in bacterial samples as described by Dehnen et al.21 All assays were carried out in the presence of NADPH (reduced β-nicotinamide adenine dinucleotide phosphate) and measured fluorometrically (excitation 460 nm, emission 517 nm) under yellow light. One milliliter of assay volume contains 100 mM triethanolamineHCl (pH 7.25), 4 mM NADPH, 60 μM benzo[a]pyrene, and 100 μL of microsomal homogenate, which was incubated for 10 min. Arbitrary fluorescence units were converted into picomoles of benzo[a]pyrenephenols formed using intercalibration between quinine sulfate and 3-hydroxybenzo[a]pyrene. The metabolite 3-hydroxybenzo[a]pyrene was obtained from the NCI Chemical Carcinogen Repository, 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 a series of grit silicon carbide papers (grades 180, 500, 800, 1200, and 1500) to a smooth surface and were finally polished to a mirror finish surface using 0.3 μm alumina powder.8 The polished coupons were rinsed with deionized water and then degreased with trichloroethylene. In the present study, system 1 consisted of 500 mL of diesel with 2% water containing 120 ppm chloride and 1% BH broth as control. Systems 2 and 3 consisted of system 1 inoculated with 2 mL of ACE2 and ACE4 as experimental systems, respectively, at about initial load of 106 CFU/mL for each system. The colony forming units (CFU) per milliliter were determined using the standard serial dilution method followed by the pour plate technique. Biocorrosion experiments were initiated by hanging pristine coupons on a nylon string in both the medium with and without the bacteria. After the incubation of the fifth and tenth days, the coupons were removed and pickled in Clark solution (2% antimony trioxide + 5% stannous chloride dissolved in concentrated HCl at room temperature with constant stirring about 510 min) and washed in water and dried with an air drier. Duplicate experiments were made for each system. Final weights of the six coupons in each system were taken, and the average corrosion rates were calculated as recommended by the National Association of Corrosion Engineers (NACE), Houston. The standard deviation for each system is presented. The pH was measured at different time periods (5 and 10 days) after the weight loss method in the presence of bacteria. A computer controlled powder X-ray diffractometer (XRD, X0 per PRO (PANalytical model)) was used to scan the corrosion products 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. The dried corrosion products were collected at the end of the incubation period, i.e., 10th day, crushed into a fine powder, and used for XRD analysis for determining the nature of oxides present in the corrosion product. The surface morphological characteristics of the control and experimental coupons API 5LX were observed under a scanning electron microscope (SEM) (Hitachi model S-3000H) at a magnification ranging from 50 to 200 operated at an accelerating voltage of 25 kV.22 10042
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Table 1. Corrosion Rate of Carbon Steel API 5LX in the Various Corrosive Systems s. no.
systems
1 500 mL diesel + 2% water containing 120 ppm chloride and 1% BH broth 2 500 mL diesel + 2% water containing120 ppm chloride and 1% BH broth + 2 mL ACE2 inoculum about 106 CFU/mL 3 500 mL diesel + 2% water containing120 ppm chloride and 1% BH broth + 2 mL ACE4 inoculum about 106 CFU/mL
2.4. Electrochemical Analysis. For electrochemical studies, a mixture of diesel oil and water (containing 120 ppm chloride ion) in the ratio 2:1 was made.23 The API 5LX steel coupon was embedded in Araldite with an exposed area of 1.0 cm2 as a working electrode. In the present study, 75 mL of 1% BH broth (containing 120 ppm chloride) and 150 mL of diesel oil were used as the control system, while 75 mL of 1% BH broth (containing 120 ppm chloride) and 150 mL of diesel 500 mL diesel inoculated with 2 mL of inoculum ACE2 and ACE4 about 104 CFU/mL were used as the experimental systems 2 and 3. The mixtures were stirred vigorously for about 120 h. After the 10th day, the coupons were removed from the respective systems and potentiodynamic polarization was carried out using potentiostate model PGP201 with volta master-1-software. A coupon of API 5LX 1 cm2 as working electrode, a standard calomel electrode (SCE) as reference electrode, and a platinum wire as counter electrode were employed for the polarization study. Tafel curves were measured with a scan rate of 0.5 mV s1 and were obtained by scanning from the open circuit potential (Ecorr) toward 200 mV anodically and 200 mV cathodically using duplicate coupons.
3. RESULTS AND DISCUSSION The total hydrocarbon at the initial stage was about 30 mg/mL in the absence of bacteria. After degradation by ACE2, the total hydrocarbon content was 10 mg/mL while in the presence of ACE4 the total hydrocarbon was 8.7 mg/mL. This may be due to the presence of higher activity of AHH in ACE4, and it further indicates that the activity of ACE4 on degradation of diesel was more severe than that of ACE2. The activities of AHH were measured during biodegradation of diesel. In the stage of biodegradation, the activity of AHH in ACE2 was measured as 11.38 p mol/min. After 20 days of bacterial inoculation in diesel, the quantity of AHH decreased significantly to below the detection limit (BDL,
