D DAVID PUBLISHING. Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors. I. Introduction

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1 Journal of Life Sciences 8 (2014) doi: / / D DAVID PUBLISHING Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors Ignatova-Ivanova Tsveteslava and Radoslav Ivanov Department of Biology, University of Shumen, Shumen 9712, Bulgaria Received: November 27, 2014 / Accepted: December 12, 2014 / Published: December 30, Abstract: Bacterial EPSs (exopolysaccharides) are believed to play an important role in the environment by promoting survival strategies such as bacterial attachment to surfaces and nutrient trapping, which facilitate processes of biofilm formation and development. These microbial biofilms have been implicated in corrosion of metals, bacterial attachment to prosthetic devices, fouling of heat exchange surfaces, toxicant immobilization, and fouling of ship hulls. In this paper, data on EPS production and the effect of EPS on corrosion of steel produced by Lactobacillus fermentum Ts are presented and discussed. The Lactobacillus fermentum Ts strain was isolated from types of Bulgarian rye flour. It was tested for its ability to produce exopolysaccharides when cultivated in a media containing 10% sucrose, 10% fructose and 10% maltose. The study of the corrosive stability of steel samples was conducted on the gravimetrique method. The rate of corrosion, the degree of protection, and coefficient of protection have been calculated. The structure of layer over steel plates was analysed by SEM (scanning electron microscopy) JSM It could be underlined that 10% sucrose and 10% maltose in the media stimulated the process of protection of corrosion. Key words: Corrosion, inhibitor, lactic acid bacteria, SEM. I. Introduction EPS (exopolysaccharide) is a term first used by Sutherland [1] to describe high-molecular-weight carbohydrate polymers produced by marine bacteria. EPSs can be found as in capsular material or as dispersed slime in the surrounding environment with no obvious association to any one particular cell [2]. Considerable progress has been made in discovering and developing new microbial EPSs that possess novel industrial significance [3]. A vast number of microbial EPSs were reported over the last decades, and their composition, structure, biosynthesis and functional properties have been extensively studied. In recent years the increased demand for natural polymers for pharmaceutical, food, and other industrial applications has led to a remarkable interest Corresponding author: Ignatova-Ivanova Tsveteslava, Ph.D., associate professor, research fields: lactic acid bacteria as probiotics products, prebiotics utilization of lactic acid bacteria, exopolysaccharides, corrosion, isolating microorganisms from Antarctica. radi_cvet@abv.bg. in polysaccharides produced by microorganisms. The physiological role of EPS depends on the ecological niches and the natural environment in which microorganisms have been isolated. Bacterial EPSs (exopolysaccharides) are believed to play an important role in the environment by promoting survival strategies such as bacterial attachment to surfaces and nutrient trapping, which facilitate processes of biofilm formation and development [4]. These microbial biofilms have been implicated in corrosion of metals, bacterial attachment to prosthetic devices, fouling of heat exchange surfaces, toxicant immobilization, and fouling of ship hulls [5-7]. Corrosion of metals is a serious and challenging problem faced worldwide by industry. It has been estimated that the yearly corrosion damage costs are currently equivalent to 4.2% of the U.S. gross national product. These costs could be greatly reduced by better and wider use of corrosion protection techniques. Traditional methods of corrosion protection involve

2 Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors 941 the use of organic coatings to protect metal surfaces through barrier and passivation mechanisms. However, these coatings are not permanent and the cost of applying organic coatings on corroding components in use is extremely prohibitive. Applying coatings before the components are introduced into service involves excessive costs because they are susceptible to abrasions and other forms of mechanically induced damage. Thus, a coating that can be easily applied and maintained on corroding parts and is cost-effective is an attractive alternative to the prevention methods currently in use. Since bacteria can coat metals with a regenerative biofilm, it is becoming evident that they may be used as a means of preventing corrosion [8]. In this paper, data on EPS production and the effect of EPS on corrosion of steel produced by Lactobacillus fermentum Ts are presented and discussed. 2. Materials and Methods 2.1 Strain Strain Lactobacillus fermentum Ts was obtained from the collection of the Department of Biology, Shumen University. Molecular analysis in LAB (lactic acid bacteria) was performed by molecular identification (16S rrna gene sequencing) in GeXP Genetic Analysis System (Beckman Coulter, USA) [9]. 2.2 Media The strain cultivated in media of MRS (de Mann Rogosa Sharpe, Biolife , Milano, Italia) in composition, g/l: Tween 80 1; pepton from casein 10.0; meat extract 8.0; yeast extract 4.0; K 2 HPO 4 2.0; sodium acetat 5.0; amonium citrate 2.0; MgSO 4 7H 2 O 0.2 and MnSO The ph of media was adjusted to 6.5 with 1 M NaOH. The basic media was sterilized by autoclaving at 121 C for 20 min, and carbohydrates supplemented were sterilized using 0.22 µm filters (Manisart ). The basic MRS broth was supplemented with 10% sucrose; 10% fructose and 10% maltose to be tested. 2.3 Study of the Corrosive Stability The study of the corrosive stability of steel samples was conducted with the gravimetrique method [10]. Before use, steel panels ( mm) were treated with 70% C 2 H 5 OH, washed with water and dried in an oven, cooled in a desiccator, weighed on a balance and kept in a desiccator unit used. The weight of the samples was measured using analytical balances. The dimensions of the samples were measured with micrometer. Three types of experimental series were performed: (a) cultivation of the studied strain in mmrs media with 10 % of sucrose; (b) in mmrs media with 10% fructose; (c) in mmrs media with 10% maltose. Initially the steel samples were added in two variants: deproteinised supernatant and free cell supernatant. Then the steel samples were added in seawater as control probe and a dilution (3: 100) of the cultural media of the studied strain was added as inhibitor of the corrosion. The duration of the procedure was 120 h at 18 C. After the treatment the steel samples were washed with water and dried to constant weight. The structure of layer over steel plates was analised by SEM (scanning electron microscopy) JSM Parameters of Corrosion After retrieval, the corrosion products were removed when washed with water. They were dried in an oven. After the removal of corrosion, steel plates were cleaned and reweighed as above to estimate weight loss. The rate of corrosion, the degree of protection, and coefficient of protection were calculated. The corrosion rate K (g/cm 2 h) was presented as follows: К = ΔG / S τ (1) Where, Δ is the corrosion rate; ΔG losses of mass consequence of corrosion, g; S is the area of plates, m 2 ; τ is duration of the corrosion, h.

942 Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors In order to track out the inhibitor properties of EPS synthesized in media, the degree of protection (Z) and coefficient of

3 942 Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors In order to track out the inhibitor properties of EPS synthesized in media, the degree of protection (Z) and coefficient of protection (γ) have been calculated using the formulas: Z = (K 0 K i ) / K 0 100, % (2) γ = K 0 / K i (3) Where, K 0 is the corrosion rate in control media; K i the corrosion rate in test media 3. Results and Discussion The corrosion of iron and its alloys causes severe economical loss resulting in a yearly cost of billions of dollars or euros. The use of heavy metals and heavy metal containing compounds, such as chromate, has to be reduced in coatings for some are known to be very toxic, even carcinogenic, and cause great environmental damage. Prevention of or reduction in the rate of corrosion may be accomplished by the use of a biological, environmentally friendly anti corrosive layer at the metal interface. The presence of EPS associated with bacterial cells can be recognized by the formation of colonies in mucous solid medium [11]. Therefore, the presence of a translucent or creamy material involving a mucoid colony is indicative of EPS production potential. When cultivated in a media with high content of saccharides such as 10% sucrose solutions, 10% fructose solutions, and 10% maltose solutions, strain L. fermentum Ts synthesizes exopolysaccharides (Fig. 1). Fig. 1 EPSs (exopolysaccharides) produced by L. fermentum Ts cultivated in a media containing 10% sucrose, which are secreted in the culture medium. Similar experiments have also been demonstrated by other authors [12, 13]. Homopolysaccharides produced by GRAS (Generally Recognised as Safe) lactic acid bacteria are often synthesised by a single extra-cellular sucrase enzyme, using only sucrose as substrate [13]. They can be produced in largest quantities (bulk scale). Moreover, their structure can be modified allowing optimisation of their physicochemical properties. By means of cyclic voltammetry, impedance measurements and potential monitoring the electrochemical behaviour of a new type of anti-corrosive biopolymers has been studied, which can be deposited upon metal surfaces as layers. Besides this electrochemical characterisation, electrochemical measurements were used to select optimal biologically manufactured and chemically modified polymers. Strain L. fermentum Ts was cultivated in a media containing 10% sucrose, 10% fructose, and 10% maltose for 12 h. The steel samples were placed in seawater as control probe and a dilution (3: 100) of the cultural media of the studied strain was added as inhibitor of the corrosion. The received results are presented in Table 1. In our previous studies [14-17], it was shown that at the presence of high concentration of lactose (5% to 15%), high concentration of sucrose 4%, mixed sucrose 4% and 2% maltose and mixed sucrose 5% and 5% maltose, mixed 5% sucrose and 5% fructose and mixed 5% sucrose and 5% fructose the strains Lactobacillus delbrueckii B5, L. delbrueckii K27, L. delbrueckii B8, L. delbrueckii O43, L. delbrueckii K3, L. delbrueckii K17, and L. delbrueckii K15 synthesized exopolysaccharides which have inhibitory properties. It is well known that some lactobacillus strains such as genus Leuconostoc secreted trans glucosidases after cultivation in the presence of sucrose. The structure of the layer over the steel plates was analyzed by Scanning electron microscopy. The results from this procedure are shown in Fig. 2.

Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors 943 Table 1 Characterization of the protective properties in seawater with added supernatant.

14 4 control 0.81 - - *The steel plates were photographed after washing; results are mean ± SEM of three separate trails. (a) (b) Fig. 2 Biofilm formed by L.

4 Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors 943 Table 1 Characterization of the protective properties in seawater with added supernatant. No The quantity of the Media sample supernatant in seawater, % K 10-5, g/cm 2 h Z, % γ 1 10% sucrose* % maltose % fructose control *The steel plates were photographed after washing; results are mean ± SEM of three separate trails. (a) (b) Fig. 2 Biofilm formed by L. delbrueckii B5 on the surface of mild steel, visualized using SEM. (a) Steel plates after corrosion in seawater with inhibitor supernatant obtained of mixed 10% sucrose; (b) control steel plates after corrosion in seawater. The biofilm makes it not easily corrodible in seawater, supplemented with cultivated ambient from the same strain grown in a composite of 10% sucrose (Fig. 2a). Fig. 2b shows a picture of a steel surface sample treated directly with seawater. The observed lamellaes are most probably FeCl 2 crystals, product of the corrosion. Microscope techniques provide information about the morphology of microbial cells and colonies, their distribution on the surface, the presence of EPS (Fig. 2a) and the nature of corrosion products (crystalline or amorphous; Fig. 2b). They can also reveal the type of attack (e.g., pitting or uniform corrosion) by visualizing changes in microstructure and surface features after removal of the biofilm and corrosion products (Fig. 2b). The ability of EPS to bind specific metal ions strongly influences its adhesion to metal surface and its ability to concentrate metal ions from surfaces and bulk media. Binding of metals may be important in both passivation and activation reactions. The observed inverse relationship between EPS and the corrosion rates of mild steel suggests that similar reactions may be occurring in the natural environment leading to the formation of a protective film on the metal surface. Biofilm [18] of a polysaccharide producing culture. Delta marina was found to act as a strong corrosion inhibitor with almost complete passivation of mild steel, reducing the corrosion rate by 95%. From this, it is evident that some microorganisms and/or their polysaccharides can act as a strong corrosion inhibitors.

5 944 Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors The corrosion of mild steel starts with generation of ferrous ions by anodic oxidation at the surface because of the reaction (Fe Fe e - ) which may undergo further oxidation producing Fe +3 species (Fe +2 Fe +3 + e - ). Ferric ions are particularly deleterious for mild steel as they tend to accelerate corrosion by the reaction (Fe +2 Fe +3 + e - ). If ferric ions are immobilized then it may be possible to control the corrosion of mild steel. Some polysaccharides are reported to exhibit the strongest stability constant for Fe 3+ ions [18]. Such a complex may serve as a corrosion inhibitor. The observed inverse relationship between EPS and the corrosion rate of mild steel suggests that such a metal-polysaccharide complex was probably involved in developing a protective film on the metal surface in natural sea water. The data suggest that biofilm EPS inhibits the corrosion of mild steel in natural marine waters. 4. Conclusions From the received results it was evident that a mixture of 10% sucrose, or 10% maltose stimulated the formation of microbial biofilm inhibiting the corrosion of steel. The present research confirms the result of the pilot project [2] that polysaccharides made by microorganisms show anti-corrosive properties. Especially, homopolysaccharides showed interesting results for the protection of steel. Measurements indicate that it takes some time for layers of biopolymers on the metal to build a complete protective layer. The data showed that L. fermentum Ts produce EPS, which serve as corrosion inhibitor for mild steel. Further studies are needed to evaluate the potential of the biofilm exopolysaccharides as anticorrosive agents. Acknowledgments The authors would like to express our gratitude for the support of this work to research grant FSI / of Shumen University. References [1] Arrage, A. A., Vasishtha, N., Sundberg, D., Bausch, G., Vincent, H. L., and White, D. C On-Line Monitoring of Antifouling and Fouling-Release Surfaces Using Bioluminescence and Fluorescence Measurements during Laminar-Flow. Journal of Industrial Microbiology 15: [2] Breur, H. J. A Fouling and Bioprotection of Metals: Monitoring and Control of Deposition Processes in Aqueous Environments. Ph.D. thesis, Technische Universiteit Delft. [3] Christensen, B. E., and Characklis, W. G Physical and chemical properties of biofilms. New York: John Wiley & Sons. [4] Costerton, W. J., Cheng, K. J., Geesey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., and Marrie, T. J Bacterial Biofilms in Nature and Disease. Annual Review of Microbiology 41: [5] Ford, T. E., Maki, J. S., and Mitchell, R Involvement of Bacterial Exopolymers in Biodeterioration. Biodeterioration 7: [6] Gómez, J Caracterización de los Exopolisacaridos Producidos por Microorganismo Shalófilos Pertenecientes a los Géneros Halomonas, Alteromonas, Idiomarina, Palleronia y Salipiger. Ph.D. thesis, Universidad de Granada. [7] Ignatova-Ivanova, Ts., Ananieva, M., Ivanov, R., Iliev, I., and Ivanova, I Biodiversity of Lactic Acid Bacteria in Bulgarian Wheat and Rye Flour. Journal of BioScience and Biotechnology [8] Ignatova-Ivanova Ts., Ivanov, R., Iliev, I., and Ivanova, I Study Anticorrosion Effect of EPS from Now Strains Lactobacillus Delbruecii. Biotechnol & Biotechnol EQ Special edition/on line [9] Ignatova-Ivanova, Ts., Ivanov, R., Iliev, I., and Ivanova, I Study of Anticorrosion Effect of Exopolysaccharides Produced Lactobacillus Delbrueckii b5 Cultivated on Different Carbohydrates. Biotechnol & Biotechnol EQ Special edition/on line [10] Ignatova-Ivanova, Ts., and Ivanov, R., Anticorrosion Effect of Biofilm Forming by Lactobacillus Strains on Metal Surfaces. Bulgarian Journal of Agricultural Science 19 (2): [11] Ignatova-Ivanova, Ts. V., and Ivanov, R. I Study of Biofilm Formed by Lactic Acid Bacteria on the Surface of Mild Steel. Journal of Life Sciences 8: doi: / [12] Jayaraman, A., Earthman, J. C., and Wood, T. K Corrosion Inhibition by Aerobic Biofilms on SAE 1018 Steel. Appl. Microbiol. Biotechnol. 47:

6 Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors 945 [13] Marshall, K. C Biofilms: an Overview of Bacterial Adhesion, Activity, and Control at Surfaces. ASM News 58: [14] Nicolaus, B., Kambourova, M., and Oner, E. T Exopolysaccharides from Extremophiles: from Fundamentals to Biotechnology. Environmental Technology 31 (10): [15] Raychev, R., Fachikov, L. and Zaprjanova, V Corrosion and Protection of the Materials Handbook for Laboratorial Exercises. Sofia. [16] Sutherland, W Bacterial Exopolysaccharides. Advances in Microbial Physiology 8: [17] Sutherland, W Biosynthesis of Microbial Exopolysaccharides. Advances in Microbial Physiology 23: [18] Geel-Schutten, G. H. van, Exopolysaccharide synthesis by Lactobacillus reuteri. Ph.D. thesis, University of Groningen.

Study of the Effect of Lactic Acid Fermentation End Products on the Speed of the Corrosion Process

ISSN: 2319-7706 Volume 4 Number 4 (2015) pp. 397-401 http://www.ijcmas.com Original Research Article Study of the Effect of Lactic Acid Fermentation End Products on the Speed of the Corrosion Process Ignatova-Ivanova