- Email: [email protected]
Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.
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4. Conclusions The microbe-immobilized membranes were directly used on oxygen electrode. The microbial biosensor response was observed by measuring the concentration of oxygen consumed by the microorganism for respiration. The biosensor showed short response time, high sensitivity and specificity. It is easy to handle and is cheaper than enzyme based biosensor. The experimental results proved that microbial biosensor based on Pseudomonas sp. is an excellent analytical tool for on-line monitoring of microbial corrosion effected by Thiobacillus sp. Because of specificity, it showed poor response in the presence of other groups of microorganisms.
Acknowledgements The financial support provided by the Department of Science and Technology, New Delhi under Young Scientist Scheme is gratefully acknowledged.
References Coleman, R.N., Gaudet, I.D., 1993. Water Res. 27, 413. Dexter, S.C. (Ed.), 1986. Biologically induced corrosion NACE-8. Proceeding of the International Conference on Biologically Induced Corrosion. National Association of Corrosion Engineer, Houston, TX. Dexter, S.C., 1993. Biofouling 7, 97. Dexter, S.C., 1996. Biofouling and biocorrosion. Bull. Electrochem. 12, 1 – 7. Dexter, S.C., Gao, G.Y., 1988. Corrosion 44, 717. Dexter, S.C., Shang H.J., 1990. Effect of biofilms, sunlight and salinity on corrosion potential and corrosion initiation of stainless alloys. EPRI NP-7275, Final Report, Project 2939–4, Electric Power Research Institute, Palo Alto, CA. Dubey, R.S., 1996. Microbiologically influenced corrosion of metallic materials and its control. Ph. Thesis, Banaras Hindu University, India.
Dubey, R.S., Upadhyay, S.N., 1999a. In: Sharma, A.S., Khanna, S.K., Sinha, A.K. (Eds.), Amperometric Microbial Biosensor for Monitoring of Microbiologically Influenced Corrosion Caused by Fungal Species. NACE/International, USA, pp. 296– 300 Also published by Akademica Books International, New Delhi, India. Dubey, R.S., Upadhyay, S.N., 1999b. A review of electrochemical techniques applied to microbiologically influenced corrosion in recent studies. Indian J. Chem. Tech. 6, 207– 218. Dubey, R.S., Mishra, R.C., Upadhyay, S.N., 1998. Isolation of aerobic microorganism and their role in microbiologically influenced corrosion of metallic materials. In: Mohandas, A., Bright Singh, I.S. (Eds.), Proceedings of the National Symposium Frontier in Applied Environmental Microbiology. Cochin, India, pp. 99 – 105. Dubey, R.S., Namboodhiri, T.K.G., Upadhyay, S.N., 1999. Microbiologically influenced corrosion of mild steel. Indian J. Chem. Tech. 2, 327– 331. Fontana, M.G., 1987. Corrosion Engineering, third ed. McGraw-Hill, New York. Jensen, A.B., Webb, C., 1995. Ferrous sulphate oxidation using Thiobacillus ferroxidans: a review. Proc. Biochem. 30 (3), 225– 236. Karube, I., Tamiya, E., Sode, K., Yokoyama, K., 1988. Application of microbiological sensors in fermentation processes. Anal. Chem. Acta 213, 69 – 77. Kurube, I., Suzuki, M., 1990. Microbial sensors. In: Cass, A.E.G. (Ed.), Biosensors: A Practical Approach. Oxford University Press, USA, pp. 327– 340. Mansfeld, F., 1988. Corros. Sci. 44, 856. Mollica, A., 1992. Int. Biodeterior. Biodegrad. 29, 52. Nakamura, K., Noike, T., Matsumoto, J., 1986. Effect of operation conditions on biological iron oxidation with rotating biological contractor. Water Res. 20, 65 – 72. Otero, T.F., Achucarro, 1994. Corrosion 50, 576. Rainina, E.I., Efremenco, E.N., Varfolomeyev, Simonian, A.L., Wilt, J.R., 1996. The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins. Biosens. Bioelectron. 11, 991– 1000. Riedel, K., Renneberg, R., Scheller, F., 1990. Adaptable microbial sensor. Anal. Lett. 23, 757– 770. Salvareza, R.C., Videla, H.A., Arvia, A.J., 1982. Corros. Sci. 22, 815. Salvareza, R.C., Videla, H.A., Arvia, A.J., 1983. Corros. Sci. 23, 717. Silverman, M.P., Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium Thiobacillus ferroxidans. J. Bacteriol. 78, 326– 331. Smith, J.R., Luthy, G.R., Middleton, A.C., 1988. Microbial ferrous iron oxidation in acid solution. J. Water Pollut. Control Fed. 60, 518– 530.
Biosensors & Bioelectronics 16 (2001) 995 – 1000 www.elsevier.com/locate/bios
Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp. R.S. Dubey *, S.N. Upadhyay Department of Chemical Engineering and Technology, Centre of Ad6anced Study, Banaras Hindu Uni6ersity, Varanasi 221005, India Received 5 July 2000; received in revised form 25 March 2001; accepted 10 April 2001
Abstract A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Immobilization; Microbial biosensor; Microbial corrosion; Pseudomonas sp.; Thiobacillus sp.
1. Introduction Over the last two decades, study of the microbiologically influenced corrosion (MIC) of metallic materials has received considerable attention (Dexter, 1986; Mollica, 1992; Dubey, 1996; Dubey et al., 1999). During the proliferation of microorganisms in an appropriate environment, various types of metabolites are produced which directly participate in electrochemical reactions at the metal/solution interface. Several investigators (Salvareza et al., 1982, 1983; Fontana, 1987; Mansfeld, 1988; Otero and Achucarro, 1994) have reported that microorganisms produce different oxidizing agents which lead to corrosion. They have also tried to explain microbial corrosion on the basis of absorption of nutrients (oxygen) during microbial growth, adherence of cells to the metal surface, liberation of corrosive metabolites or end-products, production of acids, etc. The most corrosive inorganic acid involved in the microbial corrosion is sulfuric acid, produced by * Corresponding author. Tel.: + 91-542-3172-19; fax: + 91-5423170-74. E-mail addresses: dubey – [email protected] (R.S. Dubey), [email protected] (S.N. Upadhyay).
acidophilic sulfur oxidizing bacteria (Coleman and Gaudet, 1993; Jensen and Webb, 1995). These bacteria may occur in environments where reduced sulfur compounds are present and, if oxygen is available, a very low pH (2–5) may result. Thiobacillus sp. are the chemoautotrophic bacteria, which obtain their energy for growth and other maintenance from oxidative conversion of sulfur and/or its other reduced compounds. These compounds are chemically or microbiologically oxidized into sulfate, ultimately producing highly corrosive sulfuric acid. A typical overall reaction may be written as: 4FeS2 + 15O2 + 2H2O 2Fe2(SO4)3 + 2H2SO4 The detection and confirmation of microbial corrosion is not straightforward because microbial environment and its effect on metallic corrosion processes are very complex. For analyzing the exact role of factors that influence metallic biodeterioration, online detection techniques are received to obtain quick results about redox reactions occurring at metal/environment interface. Electrochemical and other corrosion monitoring techniques give useful and quick responses when combined with biosensor technology. The application of microbial biosensors also has an edge over enzyme-
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based biosensors due to their low cost and relatively wider specificity for monitoring MIC. In the present communication, Pseudomonas sp. were isolated from microbiologically corroded metal surface and immobilized on an acetylcellulose membrane for the construction of a microbial biosensor with the help of oxygen electrode. The surface of microbe-immobilized membrane was examined by scanning electron microscope (SEM) and the effects of pH, amount of microbe and temperature were optimized. The analytical performance of this newly developed microbial biosensor was evaluated with respect to response time, sensitivity, selectivity and stability. The microbial biosensor was also used for monitoring microbial corrosion of mild steel.
Fig. 1. SEM photograph of immobilized membrane showing microorganism present on membrane surface.
2. Materials and methods
2.1. Materials All chemicals (AR grade) used in this work were purchased from Sigma (Deisenhofen, Germany) and Merck (Dermstadt, Germany). The commercial grade acetylcellulose membrane was purchased from Millipore. The Clark type oxygen electrode and Ag/AgCl electrode were purchased from Elico Co. Pvt. Ltd., Hyderabad, India.
2.2. Isolation of Pseudomonas sp. The Pseudomonas sp. were isolated from a corroded mild steel surface and were identified using standard protocols (Dubey et al., 1998). The Pseudomonas sp. were cultured under aerobic conditions at room temperature for 24 h with continuous shaking at 125 rpm in the culture medium consisting of nutrient broth granules, 6.0 g; FeSO4, 1.0 g; sodium thioglycolate, 0.01 g; methionine, 0.06 g; glutamic acid, 0.04 g; peptone, 5.0 g; yeast extract, 0.05 g and buffer solution (1 M), 50 ml/l in triple distilled water. The Pseudomonas sp. grown in this manner was used for the fabrication of microbial biosensor.
electron microscopy (SEM) for the confirmation of proper attachment of microorganism onto the surface (Fig. 1).
2.4. Construction of microbial biosensor The microbe-immobilized membrane was carefully placed on the Teflon membrane (50 mm thickness) covered tip of an oxygen electrode. The microbe retaining membrane was then covered with nylon net and held in the place with the help of an O-ring. The microbial biosensor, thus assembled, was placed into a measuring cell containing phosphate buffer and appropriate volume of test solution (aqueous sulfuric acid solution of known concentration). The schematic arrangement of the microbial biosensor is shown in Fig. 2.
2.3. Immobilization The cells of Pseudomonas sp. were harvested by centrifugation at 10,000 rpm for 15 min. The supernatant was discarded and the cells were washed three times with sterile triple distilled water. Subsequently, 2 ml of microbial cell suspension (106 spore/ml) was poured onto a porous acetylcellulose membrane (0.45 mm pore size, 4.7 mm diameter, 15 mm thickness). The microorganisms were retained over the membrane surface as an adsorbed layer. The surface of microbe-immobilized membrane was examined by scanning
Fig. 2. Scheme of microbial biosensor based on Pseudomonas sp.
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Fig. 3. Typical response curves of the microbial biosensor in 40 mM phosphate buffer containing various concentrations of sulfuric acid.
2.5. Measurement procedure Amperometric measurements were performed with a microprocessor based electrochemical interface (Gamry Instrumentation Co., USA). All experiments were carried out using a three-electrode system, with the microbial biosensor serving as working electrode. All electrochemical measurements were carried out in a 100 ml Borosil glass cell with 50 ml 0.1 M phosphate buffer solution containing the appropriate amount of sulfuric acid to provide working concentrations of 0.1, 0.4, 0.7, 1.0 and 1.2 mM at 289 0.5 °C. In each case, freshly prepared acid solution was added to the buffer solution. The temperature of the cell was maintained by an electronically controlled thermostat.
2.6. Monitoring of Thiobacillus on the metal surface The Thiobacillus sp. was cultured at various incubation periods in 9 K medium, as reported elsewhere (Silverman and Lundgren, 1959). The surface of the metal was polished sequentially with 1/0 to 4/0 grade emery paper (Jhon Okay) and washed with benzene followed by a hot analytical grade soap solution and finally with triple distilled water. After washing, samples were degreased by dipping in 99% ethanol for 30 min. The metal specimens were dried and stored over silica gel in a vacuum desiccator. The growth of Thiobacillus sp. in the culture was monitored using biocide with the help of microbial biosensor, as described above. Simultaneously, the open circuit potential (OCP) of each metal specimen exposed to Thiobacillus was measured by the corrosion potential technique (Dubey et al., 1999).
997
on immobilized Pseudomonas sp. for sulfuric acid is illustrated in Fig. 3. At the beginning, the current was obtained with buffer solution (pH 6.7, temperature 2890.5 °C) saturated with oxygen showing endogenous respiration in immobilized Pseudomonas sp. on acetylcellulose membrane. During endogenous respiration, the inherent oxygen present in situ caused oxidation of substrate present in the cytoplasm of the microbial cell and maintained equilibrium between the dissolved oxygen in medium and current level. The various concentrations of sulfuric acid (0.1, 0.4, 0.7, 1.0 and 1.1 mM) injected in the test cell containing 50 mM phosphate buffer (pH 6.7), permeated through the immobilized membrane, resulting in an increased respiration rate of microbe. Oxygen was then consumed by the microorganisms and the concentration of oxygen around the microbe-immobilized membrane decreased. The current of the microbial electrode decreased with time until a new steady state was reached which indicated that the consumption of oxygen by the microorganisms and the diffusion of oxygen from the sample solution to the membrane were in equilibrium. After each run, biosensor was removed and rinsed with triple distilled water and dipped into the freshly prepared phosphate buffer solution. The experiments were repeated several times under identical conditions in order to check the reproducibility of the data. The steady state of current revealed that the consumption of oxygen by the microorganism and diffusion of the oxygen to the immobilized bacteria from the cell containing test solution were in equilibrium, which is dependent on the concentration of sulfuric acid injected into the measuring cell. The total decrease in current was 1.82 mA and response time was : 5 min. These results are in good agreement with similar results reported by other investigators (Karube et al., 1988; Riedel et al., 1990 Kurube and Suzuki, 1990; Rainina et al., 1996)
3.2. Calibration The calibration curve (current–concentration plot) showed a linear relationship between the concentration of sulfuric acid and the decrease in current (Fig. 4). The linearity in the response of microbial biosensor was upto 0.7 mM sulfuric acid.
3.3. Reproducibility of microbial biosensor 3. Results and discussion
3.1. Response of microbial biosensor The typical response of microbial biosensor based
The reproducibility of microbial biosensor was tested by performing six sets of measurements under identical experimental conditions. The amperometric average response of microbial biosensor was reproducible within 9 0.02 mA (Table 1).
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Fig. 4. Calibration curve of the microbial biosensor in 40 mM phosphate buffer containing various concentrations of sulfuric acid.
3.4. Influence of pH and temperature The effect of pH on microbial biosensor was evaluated by varying the pH from 4 to 6 (Fig. 5). The microbial biosensor showed maximum sensitivity at nearly pH 5.2. This also corresponds to the optimal growth of Pseudomonas sp. at this pH level. The influence of temperature of microbial biosensor was also investigated and the maximum biosensor response was obtained at :28 °C (Fig. 6).
3.5. Stability of microbial biosensor The stability of microbial biosensor responses was examined at 289 5 °C in 40 mM phosphate buffer containing 0.1 mM H2SO4. The biosensor was stored at 4 °C. The stability and response are dependent upon the amount of microorganisms immobilized on membrane and incubation period. Three microbial biosensors were developed using Pseudomonas sp. cultured for different incubation periods. According to experimental data shown in Fig. 7, microbial biosensor containing bacterial cells with higher incubation period (15 days) is more stable with negligible loss in biosensor activity than those having cells of other incubation periods. Thus, experimental results reveal that a freshly pre-
Fig. 5. The effect of pH on microbial biosensor in 40 mM phosphate buffer solution containing 0.1 mM sulfuric acid.
pared culture is not essential for the fabrication of microbial biosensor.
3.6. Microbial corrosion monitoring The microbial corrosion is defined as the loss in engineering properties of the metals and its alloys as a result of metabolic activities of microorganisms. Criteria for identifying microbial corrosion problems are controversial and vary considerably with the metallic materials under test environments. Corrosion phe-
Table 1 Reproducibility of microbial biosensor in 40 mM phosphate buffer containing 0.1 mM H2SO4 Run No.
Microbial response (mA)
1 2 3 4 5 6
1.7 1.6 1.7 1.7 1.7 1.8 Fig. 6. Effect of temperature on microbial biosensor response.
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Table 2 Specificity of microbial biosensor in culture media of microorganisms
Fig. 7. Stability of microbial biosensor developed using the same amount of cell at various incubation periods (, 5 days; , 10 days; ", 15 days) in 40 mM phosphate buffer solution containing 0.1 mM sulfuric acid.
nomenon is initiated by the inhomogeneity of the environment created by the microorganisms immediately adjacent to the metal surface. This rapid process cannot be monitored quickly because of the absence of suitable techniques, which are directly applicable in the field. If biosensor and electrochemical techniques are combined together, it can play a significant role for the monitoring of microbial corrosion. In an environment enriched by Thiobacillus sp., the microorganisms produce highly corrosive sulfuric acid in presence of oxygen and water by chemical reactions. 2H2S+ 2O2 H2S2O3 +H2O 5Na2S2O3 + 4O2 +H2O 5Na2SO4 +H2SO4 +4S 4S+ 6O2 +4H2O 4H2SO4 The sulfuric acid thus produced is responsible for metallic corrosion. Several investigators (Nakamura et al., 1986; Smith et al., 1988; Dubey, 1996) have reported that both pH and oxygen concentration rapidly change during growth of Thiobacillus sp. The microbial biosensor was also tested for its response in microbiologically contaminated samples. The culture supernatant for various bacteria was exposed to the biosensor for the detection of sulfuric acid. The comparative results of the biosensor are given in Table 2. According to these data, microbial biosensor shows a very good response in Thiobacillus sp. culture medium at a very short incubation period (4 h) in comparison to other microbial environments. This means that there is a sudden change in oxygen concentration and pH due to respiratory and metabolic activities responsible for rapid influence of physico-chemical and biochemical processes of immobilized microbe on membrane at molecular level.
Microbes
Incubation periods (h)
Average response (%)
Thiobacillus sp. Desulfo6ibrio sp. E. coli A. fumigatus H. resinae
4 10 25 20 25
100 84 9 6 41 9 8 36 93 32 95
The performance of microbial electrode and its specificity were also examined. A comparison of biosensor responses for various microorganisms is shown in Table 2. The response of biosensor was treated 100% for Thiobacillus sp. Fig. 8 shows the variation of corrosion potential of mild steel exposed to the culture medium of Thiobacillus sp. It is obvious from Fig. 8 that the corrosion potential shifted in noble direction in comparison to control (pure culture medium). In the growth-monitored culture medium, the steady state of the corrosion potential is obtained after 20 min of exposure. Thus, the shift of corrosion potential towards noble direction in growth-monitored culture medium of Thiobacillus sp. suggests the protective effect for corrosion of metal. This may contribute to a good relation between biosensor response and corrosion potential. These results are in very good agreement with the results obtained by other techniques reported in the literature (Dexter and Gao, 1988; Dexter and Shang, 1990; Dexter, 1993, 1996; Dubey et al., 1999; Dubey and Upadhyay, 1999a,b). The biosensor response displayed by the Thiobacillus sp. may yield better results during microbial corrosion monitoring. Further, this method does not require expensive equipment and sample pretreatment.
Fig. 8. Variation of corrosion potential of mild steel exposed to Thiobacilius sp. at various incubation periods: (a) control (without biocide); (b) 10 h; (c) 20 h; (d) 30 h; (e) 40 h.
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4. Conclusions The microbe-immobilized membranes were directly used on oxygen electrode. The microbial biosensor response was observed by measuring the concentration of oxygen consumed by the microorganism for respiration. The biosensor showed short response time, high sensitivity and specificity. It is easy to handle and is cheaper than enzyme based biosensor. The experimental results proved that microbial biosensor based on Pseudomonas sp. is an excellent analytical tool for on-line monitoring of microbial corrosion effected by Thiobacillus sp. Because of specificity, it showed poor response in the presence of other groups of microorganisms.
Acknowledgements The financial support provided by the Department of Science and Technology, New Delhi under Young Scientist Scheme is gratefully acknowledged.
References Coleman, R.N., Gaudet, I.D., 1993. Water Res. 27, 413. Dexter, S.C. (Ed.), 1986. Biologically induced corrosion NACE-8. Proceeding of the International Conference on Biologically Induced Corrosion. National Association of Corrosion Engineer, Houston, TX. Dexter, S.C., 1993. Biofouling 7, 97. Dexter, S.C., 1996. Biofouling and biocorrosion. Bull. Electrochem. 12, 1 – 7. Dexter, S.C., Gao, G.Y., 1988. Corrosion 44, 717. Dexter, S.C., Shang H.J., 1990. Effect of biofilms, sunlight and salinity on corrosion potential and corrosion initiation of stainless alloys. EPRI NP-7275, Final Report, Project 2939–4, Electric Power Research Institute, Palo Alto, CA. Dubey, R.S., 1996. Microbiologically influenced corrosion of metallic materials and its control. Ph. Thesis, Banaras Hindu University, India.
Dubey, R.S., Upadhyay, S.N., 1999a. In: Sharma, A.S., Khanna, S.K., Sinha, A.K. (Eds.), Amperometric Microbial Biosensor for Monitoring of Microbiologically Influenced Corrosion Caused by Fungal Species. NACE/International, USA, pp. 296– 300 Also published by Akademica Books International, New Delhi, India. Dubey, R.S., Upadhyay, S.N., 1999b. A review of electrochemical techniques applied to microbiologically influenced corrosion in recent studies. Indian J. Chem. Tech. 6, 207– 218. Dubey, R.S., Mishra, R.C., Upadhyay, S.N., 1998. Isolation of aerobic microorganism and their role in microbiologically influenced corrosion of metallic materials. In: Mohandas, A., Bright Singh, I.S. (Eds.), Proceedings of the National Symposium Frontier in Applied Environmental Microbiology. Cochin, India, pp. 99 – 105. Dubey, R.S., Namboodhiri, T.K.G., Upadhyay, S.N., 1999. Microbiologically influenced corrosion of mild steel. Indian J. Chem. Tech. 2, 327– 331. Fontana, M.G., 1987. Corrosion Engineering, third ed. McGraw-Hill, New York. Jensen, A.B., Webb, C., 1995. Ferrous sulphate oxidation using Thiobacillus ferroxidans: a review. Proc. Biochem. 30 (3), 225– 236. Karube, I., Tamiya, E., Sode, K., Yokoyama, K., 1988. Application of microbiological sensors in fermentation processes. Anal. Chem. Acta 213, 69 – 77. Kurube, I., Suzuki, M., 1990. Microbial sensors. In: Cass, A.E.G. (Ed.), Biosensors: A Practical Approach. Oxford University Press, USA, pp. 327– 340. Mansfeld, F., 1988. Corros. Sci. 44, 856. Mollica, A., 1992. Int. Biodeterior. Biodegrad. 29, 52. Nakamura, K., Noike, T., Matsumoto, J., 1986. Effect of operation conditions on biological iron oxidation with rotating biological contractor. Water Res. 20, 65 – 72. Otero, T.F., Achucarro, 1994. Corrosion 50, 576. Rainina, E.I., Efremenco, E.N., Varfolomeyev, Simonian, A.L., Wilt, J.R., 1996. The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins. Biosens. Bioelectron. 11, 991– 1000. Riedel, K., Renneberg, R., Scheller, F., 1990. Adaptable microbial sensor. Anal. Lett. 23, 757– 770. Salvareza, R.C., Videla, H.A., Arvia, A.J., 1982. Corros. Sci. 22, 815. Salvareza, R.C., Videla, H.A., Arvia, A.J., 1983. Corros. Sci. 23, 717. Silverman, M.P., Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium Thiobacillus ferroxidans. J. Bacteriol. 78, 326– 331. Smith, J.R., Luthy, G.R., Middleton, A.C., 1988. Microbial ferrous iron oxidation in acid solution. J. Water Pollut. Control Fed. 60, 518– 530.
Biosensors & Bioelectronics 16 (2001) 995 – 1000 www.elsevier.com/locate/bios
Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp. R.S. Dubey *, S.N. Upadhyay Department of Chemical Engineering and Technology, Centre of Ad6anced Study, Banaras Hindu Uni6ersity, Varanasi 221005, India Received 5 July 2000; received in revised form 25 March 2001; accepted 10 April 2001
Abstract A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Immobilization; Microbial biosensor; Microbial corrosion; Pseudomonas sp.; Thiobacillus sp.
1. Introduction Over the last two decades, study of the microbiologically influenced corrosion (MIC) of metallic materials has received considerable attention (Dexter, 1986; Mollica, 1992; Dubey, 1996; Dubey et al., 1999). During the proliferation of microorganisms in an appropriate environment, various types of metabolites are produced which directly participate in electrochemical reactions at the metal/solution interface. Several investigators (Salvareza et al., 1982, 1983; Fontana, 1987; Mansfeld, 1988; Otero and Achucarro, 1994) have reported that microorganisms produce different oxidizing agents which lead to corrosion. They have also tried to explain microbial corrosion on the basis of absorption of nutrients (oxygen) during microbial growth, adherence of cells to the metal surface, liberation of corrosive metabolites or end-products, production of acids, etc. The most corrosive inorganic acid involved in the microbial corrosion is sulfuric acid, produced by * Corresponding author. Tel.: + 91-542-3172-19; fax: + 91-5423170-74. E-mail addresses: dubey – [email protected] (R.S. Dubey), [email protected] (S.N. Upadhyay).
acidophilic sulfur oxidizing bacteria (Coleman and Gaudet, 1993; Jensen and Webb, 1995). These bacteria may occur in environments where reduced sulfur compounds are present and, if oxygen is available, a very low pH (2–5) may result. Thiobacillus sp. are the chemoautotrophic bacteria, which obtain their energy for growth and other maintenance from oxidative conversion of sulfur and/or its other reduced compounds. These compounds are chemically or microbiologically oxidized into sulfate, ultimately producing highly corrosive sulfuric acid. A typical overall reaction may be written as: 4FeS2 + 15O2 + 2H2O 2Fe2(SO4)3 + 2H2SO4 The detection and confirmation of microbial corrosion is not straightforward because microbial environment and its effect on metallic corrosion processes are very complex. For analyzing the exact role of factors that influence metallic biodeterioration, online detection techniques are received to obtain quick results about redox reactions occurring at metal/environment interface. Electrochemical and other corrosion monitoring techniques give useful and quick responses when combined with biosensor technology. The application of microbial biosensors also has an edge over enzyme-
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based biosensors due to their low cost and relatively wider specificity for monitoring MIC. In the present communication, Pseudomonas sp. were isolated from microbiologically corroded metal surface and immobilized on an acetylcellulose membrane for the construction of a microbial biosensor with the help of oxygen electrode. The surface of microbe-immobilized membrane was examined by scanning electron microscope (SEM) and the effects of pH, amount of microbe and temperature were optimized. The analytical performance of this newly developed microbial biosensor was evaluated with respect to response time, sensitivity, selectivity and stability. The microbial biosensor was also used for monitoring microbial corrosion of mild steel.
Fig. 1. SEM photograph of immobilized membrane showing microorganism present on membrane surface.
2. Materials and methods
2.1. Materials All chemicals (AR grade) used in this work were purchased from Sigma (Deisenhofen, Germany) and Merck (Dermstadt, Germany). The commercial grade acetylcellulose membrane was purchased from Millipore. The Clark type oxygen electrode and Ag/AgCl electrode were purchased from Elico Co. Pvt. Ltd., Hyderabad, India.
2.2. Isolation of Pseudomonas sp. The Pseudomonas sp. were isolated from a corroded mild steel surface and were identified using standard protocols (Dubey et al., 1998). The Pseudomonas sp. were cultured under aerobic conditions at room temperature for 24 h with continuous shaking at 125 rpm in the culture medium consisting of nutrient broth granules, 6.0 g; FeSO4, 1.0 g; sodium thioglycolate, 0.01 g; methionine, 0.06 g; glutamic acid, 0.04 g; peptone, 5.0 g; yeast extract, 0.05 g and buffer solution (1 M), 50 ml/l in triple distilled water. The Pseudomonas sp. grown in this manner was used for the fabrication of microbial biosensor.
electron microscopy (SEM) for the confirmation of proper attachment of microorganism onto the surface (Fig. 1).
2.4. Construction of microbial biosensor The microbe-immobilized membrane was carefully placed on the Teflon membrane (50 mm thickness) covered tip of an oxygen electrode. The microbe retaining membrane was then covered with nylon net and held in the place with the help of an O-ring. The microbial biosensor, thus assembled, was placed into a measuring cell containing phosphate buffer and appropriate volume of test solution (aqueous sulfuric acid solution of known concentration). The schematic arrangement of the microbial biosensor is shown in Fig. 2.
2.3. Immobilization The cells of Pseudomonas sp. were harvested by centrifugation at 10,000 rpm for 15 min. The supernatant was discarded and the cells were washed three times with sterile triple distilled water. Subsequently, 2 ml of microbial cell suspension (106 spore/ml) was poured onto a porous acetylcellulose membrane (0.45 mm pore size, 4.7 mm diameter, 15 mm thickness). The microorganisms were retained over the membrane surface as an adsorbed layer. The surface of microbe-immobilized membrane was examined by scanning
Fig. 2. Scheme of microbial biosensor based on Pseudomonas sp.
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Fig. 3. Typical response curves of the microbial biosensor in 40 mM phosphate buffer containing various concentrations of sulfuric acid.
2.5. Measurement procedure Amperometric measurements were performed with a microprocessor based electrochemical interface (Gamry Instrumentation Co., USA). All experiments were carried out using a three-electrode system, with the microbial biosensor serving as working electrode. All electrochemical measurements were carried out in a 100 ml Borosil glass cell with 50 ml 0.1 M phosphate buffer solution containing the appropriate amount of sulfuric acid to provide working concentrations of 0.1, 0.4, 0.7, 1.0 and 1.2 mM at 289 0.5 °C. In each case, freshly prepared acid solution was added to the buffer solution. The temperature of the cell was maintained by an electronically controlled thermostat.
2.6. Monitoring of Thiobacillus on the metal surface The Thiobacillus sp. was cultured at various incubation periods in 9 K medium, as reported elsewhere (Silverman and Lundgren, 1959). The surface of the metal was polished sequentially with 1/0 to 4/0 grade emery paper (Jhon Okay) and washed with benzene followed by a hot analytical grade soap solution and finally with triple distilled water. After washing, samples were degreased by dipping in 99% ethanol for 30 min. The metal specimens were dried and stored over silica gel in a vacuum desiccator. The growth of Thiobacillus sp. in the culture was monitored using biocide with the help of microbial biosensor, as described above. Simultaneously, the open circuit potential (OCP) of each metal specimen exposed to Thiobacillus was measured by the corrosion potential technique (Dubey et al., 1999).
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on immobilized Pseudomonas sp. for sulfuric acid is illustrated in Fig. 3. At the beginning, the current was obtained with buffer solution (pH 6.7, temperature 2890.5 °C) saturated with oxygen showing endogenous respiration in immobilized Pseudomonas sp. on acetylcellulose membrane. During endogenous respiration, the inherent oxygen present in situ caused oxidation of substrate present in the cytoplasm of the microbial cell and maintained equilibrium between the dissolved oxygen in medium and current level. The various concentrations of sulfuric acid (0.1, 0.4, 0.7, 1.0 and 1.1 mM) injected in the test cell containing 50 mM phosphate buffer (pH 6.7), permeated through the immobilized membrane, resulting in an increased respiration rate of microbe. Oxygen was then consumed by the microorganisms and the concentration of oxygen around the microbe-immobilized membrane decreased. The current of the microbial electrode decreased with time until a new steady state was reached which indicated that the consumption of oxygen by the microorganisms and the diffusion of oxygen from the sample solution to the membrane were in equilibrium. After each run, biosensor was removed and rinsed with triple distilled water and dipped into the freshly prepared phosphate buffer solution. The experiments were repeated several times under identical conditions in order to check the reproducibility of the data. The steady state of current revealed that the consumption of oxygen by the microorganism and diffusion of the oxygen to the immobilized bacteria from the cell containing test solution were in equilibrium, which is dependent on the concentration of sulfuric acid injected into the measuring cell. The total decrease in current was 1.82 mA and response time was : 5 min. These results are in good agreement with similar results reported by other investigators (Karube et al., 1988; Riedel et al., 1990 Kurube and Suzuki, 1990; Rainina et al., 1996)
3.2. Calibration The calibration curve (current–concentration plot) showed a linear relationship between the concentration of sulfuric acid and the decrease in current (Fig. 4). The linearity in the response of microbial biosensor was upto 0.7 mM sulfuric acid.
3.3. Reproducibility of microbial biosensor 3. Results and discussion
3.1. Response of microbial biosensor The typical response of microbial biosensor based
The reproducibility of microbial biosensor was tested by performing six sets of measurements under identical experimental conditions. The amperometric average response of microbial biosensor was reproducible within 9 0.02 mA (Table 1).
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Fig. 4. Calibration curve of the microbial biosensor in 40 mM phosphate buffer containing various concentrations of sulfuric acid.
3.4. Influence of pH and temperature The effect of pH on microbial biosensor was evaluated by varying the pH from 4 to 6 (Fig. 5). The microbial biosensor showed maximum sensitivity at nearly pH 5.2. This also corresponds to the optimal growth of Pseudomonas sp. at this pH level. The influence of temperature of microbial biosensor was also investigated and the maximum biosensor response was obtained at :28 °C (Fig. 6).
3.5. Stability of microbial biosensor The stability of microbial biosensor responses was examined at 289 5 °C in 40 mM phosphate buffer containing 0.1 mM H2SO4. The biosensor was stored at 4 °C. The stability and response are dependent upon the amount of microorganisms immobilized on membrane and incubation period. Three microbial biosensors were developed using Pseudomonas sp. cultured for different incubation periods. According to experimental data shown in Fig. 7, microbial biosensor containing bacterial cells with higher incubation period (15 days) is more stable with negligible loss in biosensor activity than those having cells of other incubation periods. Thus, experimental results reveal that a freshly pre-
Fig. 5. The effect of pH on microbial biosensor in 40 mM phosphate buffer solution containing 0.1 mM sulfuric acid.
pared culture is not essential for the fabrication of microbial biosensor.
3.6. Microbial corrosion monitoring The microbial corrosion is defined as the loss in engineering properties of the metals and its alloys as a result of metabolic activities of microorganisms. Criteria for identifying microbial corrosion problems are controversial and vary considerably with the metallic materials under test environments. Corrosion phe-
Table 1 Reproducibility of microbial biosensor in 40 mM phosphate buffer containing 0.1 mM H2SO4 Run No.
Microbial response (mA)
1 2 3 4 5 6
1.7 1.6 1.7 1.7 1.7 1.8 Fig. 6. Effect of temperature on microbial biosensor response.
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Table 2 Specificity of microbial biosensor in culture media of microorganisms
Fig. 7. Stability of microbial biosensor developed using the same amount of cell at various incubation periods (, 5 days; , 10 days; ", 15 days) in 40 mM phosphate buffer solution containing 0.1 mM sulfuric acid.
nomenon is initiated by the inhomogeneity of the environment created by the microorganisms immediately adjacent to the metal surface. This rapid process cannot be monitored quickly because of the absence of suitable techniques, which are directly applicable in the field. If biosensor and electrochemical techniques are combined together, it can play a significant role for the monitoring of microbial corrosion. In an environment enriched by Thiobacillus sp., the microorganisms produce highly corrosive sulfuric acid in presence of oxygen and water by chemical reactions. 2H2S+ 2O2 H2S2O3 +H2O 5Na2S2O3 + 4O2 +H2O 5Na2SO4 +H2SO4 +4S 4S+ 6O2 +4H2O 4H2SO4 The sulfuric acid thus produced is responsible for metallic corrosion. Several investigators (Nakamura et al., 1986; Smith et al., 1988; Dubey, 1996) have reported that both pH and oxygen concentration rapidly change during growth of Thiobacillus sp. The microbial biosensor was also tested for its response in microbiologically contaminated samples. The culture supernatant for various bacteria was exposed to the biosensor for the detection of sulfuric acid. The comparative results of the biosensor are given in Table 2. According to these data, microbial biosensor shows a very good response in Thiobacillus sp. culture medium at a very short incubation period (4 h) in comparison to other microbial environments. This means that there is a sudden change in oxygen concentration and pH due to respiratory and metabolic activities responsible for rapid influence of physico-chemical and biochemical processes of immobilized microbe on membrane at molecular level.
Microbes
Incubation periods (h)
Average response (%)
Thiobacillus sp. Desulfo6ibrio sp. E. coli A. fumigatus H. resinae
4 10 25 20 25
100 84 9 6 41 9 8 36 93 32 95
The performance of microbial electrode and its specificity were also examined. A comparison of biosensor responses for various microorganisms is shown in Table 2. The response of biosensor was treated 100% for Thiobacillus sp. Fig. 8 shows the variation of corrosion potential of mild steel exposed to the culture medium of Thiobacillus sp. It is obvious from Fig. 8 that the corrosion potential shifted in noble direction in comparison to control (pure culture medium). In the growth-monitored culture medium, the steady state of the corrosion potential is obtained after 20 min of exposure. Thus, the shift of corrosion potential towards noble direction in growth-monitored culture medium of Thiobacillus sp. suggests the protective effect for corrosion of metal. This may contribute to a good relation between biosensor response and corrosion potential. These results are in very good agreement with the results obtained by other techniques reported in the literature (Dexter and Gao, 1988; Dexter and Shang, 1990; Dexter, 1993, 1996; Dubey et al., 1999; Dubey and Upadhyay, 1999a,b). The biosensor response displayed by the Thiobacillus sp. may yield better results during microbial corrosion monitoring. Further, this method does not require expensive equipment and sample pretreatment.
Fig. 8. Variation of corrosion potential of mild steel exposed to Thiobacilius sp. at various incubation periods: (a) control (without biocide); (b) 10 h; (c) 20 h; (d) 30 h; (e) 40 h.