Abbreviations: Sulfate-Reducing Bacteria (SRB); scanning electronic microscope (SEM); Energy Dispersive X-ray spectrometry (EDS)
The oil industry is an important driving force of the Algerian economy. However, this industry is faced with numerous challenges. One of these challenges is bacterial corrosion. Corrosion, in general, can be defined as a phenomenon of materials degradation under the effect of the physical, chemical or electrochemical actions of the surrounding environment. Corrosion is responsible for annual costs up to 5% of the Gross National Product (GNP) of an industrial country .
The process where bacterial metabolic acts help creating favorable conditions for corrosion to occur is called bacterial corrosion . The main bacterial type associated with this deterioration is the anaerobic Sulfate-Reducing Bacteria (SRB). In low oxygen environments and in the presence of sulfate, SRBs contribute to the mineralization of organic material by the process of sulfate reduction [4-7]. Furthermore, these bacteria are known for their exceptional enzymatic capability that allows them to grow in rather complex conditions and to adhere to various surfaces, including metallic based materials [8-10]. This bacterial corrosion is widely observed in petroleum pipelines which might result in pipes clogging and other severe losses in the oil and gas industries .
In order to avoid the harmful effects of BSR on metallic materials and installations, a chemical treatment with biocides is applied. The term biocide or bactericide includes all the chemicals which destroy or prevent the development of microorganisms . Quaternary ammonium compounds (QUATS), formaldehyde, glutaraldehyde, acrolein, amines and diamines in addition to methylchloromethyl-isothiazolone (MCMI) and 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOI) are some of the widely used compounds in industrial biocides .
The purpose of this work was to study the deposit resulting from bacterial corrosion on the surface of N80 steel samples and determine the elements contributing to the formation of this deposit. Furthermore, this study evaluated the effectiveness of the industrial biocide 2 CHIMEC 7660 in the removal of these deposits.
The test material in this work was N80 steel which is known for its high resistance to the pressure. This has rendered it a proper material to be used during the drilling process and to ensure a normal functionality in oil wells especially in early extraction phases.
The used biocide is a commercial substance under the name Biocide2 CHIMEC 7660 (CHIMEC – Rome). This biocide is a liquid, non-oxidant, non-foaming substance with an antibacterial effect against aerobic and anaerobic bacteria which make it suitable for use on hard water and over a wide range of pH values (6-9).
A sample of water was collected from CINA (North Industrial Complex Treatment Center), 9 km north of Hassi Messaoud (South Algeria). This sample of water comes from crude separation. To examine the presence of (SRB) in this sample, a bacterial culture medium (SRB specific)  was prepared (Table 1). Penicillin vials were filled with 9 ml of the prepared medium with a metal nail. The vials were plugged using proper rubber plugs, purged with nitrogen to create an anaerobic environment, and sterilized by autoclaving at 120°C for 50 minutes. Then, using a syringe, 1 ml of the water sample was injected through the septum of each vial. Then, the vials were incubated at 37 °C for 28 days. By the end of the incubation period, a blackening deposit formed in the vials with an increase in environment pH which illustrated the presence of (SRB) in the water sample.
Table 1. The chemical composition of the culture environment of bacteria
N80 oil steel pipe samples of 15 mm*13.5 mm* 5.4 mm for length, weight and thickness respectively, and with the chemical composition showed in (Table 2) were used in the experiment. The surface of the samples was rectified, cleaned, and dried.
Table 2. The chemical composition of the base metal (weight %)
The same bacterial culture environment shown in (Table 1) was prepared. Penicillin vials were filled with 9 ml of the prepared environment in addition to the N80 steel sample in each vial. The vials were airtight sealed, purged with nitrogen, and sterilized by autoclaving at 120 °C for 50 minutes. Then, 1 ml of the (SRB) water sample was injected through the septum of each vial.
The vials containing the samples were incubated 37 °C for 90 days (Control) or treated after 60 days of incubation by injecting 1 ml of Biocide 2 CHIMEC 7660 (CHIMEC – Rome) and left in the incubator at 37°C for another 30 days (Table 3). Medium pH was measured before and after incubation for both control and treatment using PHS-3E pH Meter
Table 3. Description of the samples
Samples surfaces were inspected using scanning electronic microscope (SEM) to determine the morphological aspects of each sample surface with magnifications of X65, X500, and X1000. Furthermore, Quanta SEM-EDS analytical system was used in order to determine the elements that contribute to the formation of this deposit.
Results showed that the pH of the Control environment was increased from 7.1 before incubation to 9.01 after incubation, forming a more preferable environment for bacteria and thereafter, formation of biofilm clusters which can develop even under extreme conditions of temperature and hydrostatic pressure . On the other hand, a decrease in pH (4.89) was observed in biocide treated sample. This low pH medium can trigger an inhibitory effect on bacterial growth with the presence of H2S .
The morphological aspects of the formed deposits were inspected using scanning electron microscopy (Fig. 1). The surface of the control sample clearly demonstrated the existence of a stable adherent deposit in the form of colonies (Fig. 1 A, B and C); whereas large cleared areas were observed in the surface of Biocide treated sample (Fig. 1 D, E, and F). This indicates the high efficiency of the used Biocide in removing these deposits and preventing bacterial aggregation.
In order to understand the chemical compositions of the layer formed on N80 samples surfaces, Energy Dispersive X-ray spectrometry was used. The obtained maps were used to analyze chemical elements distribution with an allocation of color for each detected element (Fig. 2). Maps analysis results showed that the elements existed in the deposit layer were: Oxygen (O) which is here a result of water ionization, Iron (Fe) as a result of iron oxidation, Carbon (C), Calcium (Ca), Magnesium (Mg), Phosphorus (P), and Sulfur (S) which resulted from the conversion of Magnesium Sulfate and Ammonium Sulfate . Sulfate-Reducing Bacteria efficiently use the compounds derived from the oxidation of sulfur as electron acceptors. These compounds such as Sulfate (SO4-2), Sulfites (SO3-2), Thiosulfates (S2O4-2), and elemental Sulfur (S°) are reduced entirely to Sulfide . The biochemical reactions involved in SRB activities end with iron oxide and iron Sulfide  which explains the high iron oxide and Sulfide in the formed deposit.
Figure 2. EDS elemental maps of N80 steel samples incubated in SRB contaminated anaerobic environment for 90 days at 37 °C (control) (First raw) and incubated in SRB contaminated anaerobic environment for 60 days at 37 °C then treated by biocide2 CHIMEC 7660 and left for another 30 days at 37 °C (Treatment) (Second raw). Scale bars represent 100 µm in all maps. The numbers next to each map refer to element distribution regions of interest (ROIs). The lighter colored image for each element refers to a higher concentration for this element in the studied area.
EDS spectrum analysis for the treated sample illustrated that the main surface deposits were O, Fe, and C with 98.9% in T2 scan and 100% in T1 scan for the three elements combined with only traces of S and K; while this ratio did not exceed 78.8% for the three elements combined in control sample with 22% and 21.2% Mg, Ca, P, and S combination in C1 and C2 respectively (Fig. 3). Phosphorus and sulfur precipitates are usually interpreted as an indicator for SRB activity  which refers to the increased activity in the non-protected steel sample in comparison to the biocide protected sample.
Figure 3. EDS analysis spectrum and the chemical composition for the deposits formed on N80 steel samples incubated in SRB contaminated anaerobic environment for 90 days at 37 °C (Control) (C1 and C2), and incubated in SRB contaminated anaerobic environment for 60 days at 37 °C then treated by biocide2 CHIMEC 7660 and left for another 30 days at 37 °C (Treatment) (T1 and T2).
Biocide treatment lowered pH levels for the environment rendering it unfavorable for SRB growth and biofilm-forming in comparison to the control sample.
Corrosion signs were observed in both biocides treated and untreated samples; therefore, 60 days of exposure to SRB are enough to develop bacterial corrosion in untreated N80 steel.
Although Biocide application was efficient enough to control bacterial growth and eliminate the forming residues after 60 days of exposure to (SRB), the bacterial corrosion occurred before the treatment was enough to cause damage to the studied surface which refers to the effectiveness of using Biocide 2 CHIMEC 7660 and emphasizes on its early application.