BIOFILM FORMATION ON BRASS COUPONS EXPOSED TO COOLING WATER

BIOFILM FORMATION ON BRASS COUPONS EXPOSED TO COOLING WATER

M.T.S. Lutterbach¹ and F.P. de França2

¹Instituto de Microbiologia / Universidade Federal do Rio de Janeiro (UFRJ) - Rio de Janeiro, RJ - Brazil

²Departamento de Engenharia Bioquímica Escola de Química - Universidade Federal do Rio de Janeiro

Centro de Tecnologia, Bloco E, Ilha do Fundão - Rio de Janeiro, RJ - 21941 900 - Brazil

Phone: [phone] - Fax: [phone]

(Received: August 6, 1996; Accepted: January 30, 1997)

Abstract - Brass coupons were installed in a bypass in an industrial cooling water that uses seawater. The metal samples were removed at 15, 30, 45, and 60-day intervals for quantitative and qualitative analyses of the microorganisms constituting the biofilm adhering to the metal surface. After 15 days of exposure, a biofilm had already been generated which contained aerobic and anaerobic microorganisms. The aerobic bacteria were the most stable in relation to quantity, followed by the fungi. Anaerobic microorganisms, as well as sulfate-reducing bacteria, were present at higher concentrations. Variations in sulfide contents were observed in the biofilm. Analysis by scanning electron microscopy revealed microalgae, bacteria, filaments, and corrosion products as constituents of the biofilm adhering to the surface of the metal. After the biofilms were scraped off the brass samples, evidence of corrosion was observed on the metal surface.

Keywords: Biofilms, brass, cooling water, seawater.

INTRODUCTION

For many years, microbiologically-influenced corrosion (MIC) was regarded with a certain discredit. Nowadays, however, MIC control is universally accepted and recognized as an ample area of study in the prevention of corrosion processes which could otherwise lead to great financial losses (Stoecker II, 1994).

The literature includes numerous case histories involving MIC in the most varied industrial plants, such as wastewater treatment (Soebbing and Yolo, 1995), oil field water injection (Elboujdaini and Sastri, 1995), and water systems of a nuclear power plant (Licina, 1990; Zisson et al., 1995), among others.

Industrial systems possess a number of characteristics that favor the formation of biofilms, and, therefore, MIC. Stagnant water, untreated water, and intermittent flow rates are among the most common examples (Licina, 1990).

Biofilms can develop in different shapes, depending on the chemical characteristics of the water and the dynamics of the system; the structure of the biofilm, in turn, has a direct influence upon the mechanisms of corrosion. Biofilm-forming communities are subject to variations that depend upon environmental parameters (Lee et al., 1995).

Seeking to understand biofilm structure and activity and its relation to corrosion, Lewandowski et al. (1995) observed that some cases of MIC can be related to the heterogeneity of the biofilm. Presenting the authors define biofilms as complex structures made up of cell clusters (aggregates of bacterial cells in an exopolysaccharides (EPS) matrix ) and interstitial spaces, contrary to the traditional concept of a planar structure with homogeneous cell distribution.

The formation of a biofilm takes place when a solid surface comes into contact with a liquid medium. Organic substances and minerals are transported to the surface and create a conditioning film, where nutrients are concentrated and allow the replication of microorganisms present in the aqueous environment (Characklis and Marshall, 1990).

Since metal surfaces covered with biofilms are subject to corrosion, and the microorganisms comprising this biofilm accelerate the corrosion process, the aim of the work was to evaluate the biofilm formed on brass coupons exposed to industrial cooling water and to determine whether or not microbiologically influenced corrosion occurred in the metal samples.

MATERIALS AND METHODS

Field Experiments

The experiments were conducted at the Duque de Caxias Refinery (REDUC)- Petrobrás, located on the Guanabara Bay in the city of Rio de Janeiro, Brazil.

REDUC uses seawater in heat exchangers. Since this water is frequently used without treatment, its composition is subject to variations due to contamination with waste from neighboring industrial plants. The metal samples used for testing were made of brass, with an area of 1.78 cm², and they were installed in a bypass set up in one of the plant’s cooling towers.

During the experiments, the mean water flow was 2.4 m³ / h. The temperature was maintained at 29 + 2 ºC, and the pH was 7.8.

The brass coupons were removed from the cooling water for analysis at 15, 30, 45, and 60-day intervals.

Preparation of Suspensions for Analysis

After being withdrawn from the salt water, the brass samples were rinsed with sterile distilled water containing 2% NaCl in order to remove planctonic microorganisms.

The suspension for analysis of aerobic microorganisms was obtained by scraping off one of the surfaces of the metal with a sterile spatula under aseptic conditions, as described by Cook and Gaylarde (1988). Biofilm samples were then suspended in 30 ml of a 2% NaCl solution.

The same technique was applied for analysis of anaerobic microorganisms, by substituting the NaCl solution with a reducing solution: NaCl 8,0 g; sodium thioglycollate 0,124 g; ascorbic acid 0,1 g; resazurine 0,0025 g; destiled water 1000 ml, (Postgate, 1984) previously degassed with N₂ ( Lutterbach and de França, 1996).

Quantitative Determinations

Quantification of aerobic bacteria

Nutrient broth (Merck) was used as culture medium and the number of bacteria were quantified according to the technique of the most probable number (MPN) (Koch, 1981) at 32ºC ± 1ºC for 48 hours.

Quantification of anaerobic bacteria

The MPN technique was applied and the culture medium was thioglycollate fluid (Difco). The tests were conducted under anaerobic conditions at 32ºC ± 1ºC for 28 days.

Quantification of sulfate-reducing bacteria (SRB)

The technique was essentially the same as above, substituting Postgate B medium (Postgate, 1984) as the culture medium.

Quantification of fungi

These were quantified by enumeration of colony forming units (cfu) on Petri dishes containing Sabouraud medium (Merck). Incubation occurred at 25ºC ± 1ºC for seven days.

Quantification of total sulfides

The colorimetric method according to APHA (1972) was used. The standard curve used for reference was obtained with Na₂ S - monohydrate P.A. (Merck). Readings were taken at 670 nm.

Qualitative Determinations

Samples of the suspensions prepared for analysis of aerobic microorganisms were inoculated into a culture medium recommended by APHA (1992) in order to verify the presence of bacteria of the genus Gallionella. The same microbial suspensions were also grown in a modified Chú medium (Rosemberg, 1976) for analysis of microalgae.

Laboratory Experiments

In order to verify the effect of seawater upon the metal, brass samples were placed in jars containing 100 ml of the same water used in the bypass. This system was sterilized at 121ºC for twenty minutes (de França and Lutterbach, 1996).

Analysis by Scanning Electron Microscopy (SEM)

SEM analysis was carried out on coupons containing biofilms after exposure to seawater. Specimens were fixed immediately after in situ collection with 2.5% glutaraldehyde solution and 0.1M cacodylate buffer, 1:1 (vol:vol) in sea water, for 24 h at 4^oC. Next, samples were first rinsed with 0.1M cacodylate buffer and subsequently with decreasing concentrations of sea water (30%, 20%, 5%, tridistilled water) for desalination. Samples were then dehydrated through an acetone series to 100% (30%, 50%, 70%, 90%, 100%). The samples were then dried by injection of CO₂ in a critical point drying apparatus CPD-030 Balzers (Coutinho et al. 1993). The dried samples then received a layer of gold with a Balzers Union SCD - 040 and were observed through the Jeol JXA 840 Electron Probe Microanalyzer.

Identification of bacteria

The most frequent aerobic bacteria were identified according to Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994).

RESULTS AND DISCUSSION

After fifteen days of exposure, the metal coupons already presented completely formed biofilms with large variations of microbial constituents. According to Characklis and Marshall (1990), biofilm formation begins once a surface is immersed in a liquid medium, through the generation of a conditioning film. After fifteen days of exposure (Table 1), the biofilme formed contained anaerobic and aerobic microorganisms. The aerobic microorganisms are the ones that create the anaerobic environment through microbial growth, metabolic products, and consumption of oxygen, as well as by limiting the diffusion of oxygen across the biofilm layers.

During the period of the experiment, the number of bacteria aerobic cells were most constant, followed by the fungi. The anaerobic microorganisms, however, were present in increased concentrations. It is worth noting that the concentration of sulfate fell after thirty days, probably as a result of the sloughing of tubercles from the metal surface due to water flow.

The relation of sulfide content and the number of SRB cells it was not detected, perhaps to the fact that the biofilm is a dynamic structure which detaches and renews itself in time. All that can be affirmed is that SRB are present and active in this environment.

Bacteria of the genus Gallionella were detected throughout the experimental period. These are frequently cited in the literature as being associated to SRB in biofilms due they contribute to the criation of anaerobic environment and their metabolic activity Fe biooxidans, that favor the development of this microorganism (NACE, 1976).

Biofilm constituents* 15 days 30 days 45 days 60 days aerobic bacteria cells/cm2 fungi cfu/cm2 anaerobic bacteria cells/cm2 SRB cells/cm2Gallionellaalgae sulfide mcg/cm2 7.7 + 0.7 x 103 4.0 + 0.5 x 103 3.5 + 1.0 x 105 7.5 + 0.8 x 102 (+) (+) 1412.53 1.3 + 0.9 x 104 7.1 + 0.8 x 10 1.3 + 0.5 x 104 6.0 + 1.0 x 103 (+) (-) 485.89 9.7 + 1.0 x 103 2.3 + 0.8 x 102 9.2 + 0.9 x 105 4.0 + 0.4 x 105 (+) (-) 81.43 2.0 +0.8 x 104 6.7 + 0.7 x 10 9.0 + 0.5 x 1010 2.0 + 0.5 x 105 (+) (-) 57.89

Biofilm constituents*	15 days	30 days	45 days	60 days
aerobic bacteria cells/cm2 fungi cfu/cm2 anaerobic bacteria cells/cm2 SRB cells/cm2 Gallionella algae sulfide mcg/cm2	7.7 + 0.7 x 103 4.0 + 0.5 x 103 3.5 + 1.0 x 105 7.5 + 0.8 x 102 (+) (+) 1412.53	1.3 + 0.9 x 104 7.1 + 0.8 x 10 1.3 + 0.5 x 104 6.0 + 1.0 x 103 (+) (-) 485.89	9.7 + 1.0 x 103 2.3 + 0.8 x 102 9.2 + 0.9 x 105 4.0 + 0.4 x 105 (+) (-) 81.43	2.0 +0.8 x 104 6.7 + 0.7 x 10 9.0 + 0.5 x 1010 2.0 + 0.5 x 105 (+) (-) 57.89

Table 1: Microorganisms and sulfides detected in the biofilms that formed on the brass coupons exposed to seawater. Mean value of four experiments

(+) present; (-) absent

The most frequently found aerobic bacteria in the biofilms were isolated and identified as Pseudomonas putrefaciens and Bacillus sp. These are often associated with microfouling, as the genus Pseudomonas produces EPS, one of the agents responsible for trapping other organisms in the biofilm, thereby adding to biofilm thickness (Page and Gaylarde, 1990).

Fifteen days after the start of this experiment, the presence of algae was observed in the biofilms. Different shaped algae of the Diatomaceae group were detected through SEM (Figure 1). Figures 2 and 3 demonstrate that some of the algae acted as supports for smaller cells - in this case, bacteria. This is due to the nutritional interaction between the two, since algae consume carbon dioxide produced by the bacteria, producing in turn organic carbon and oxygen that are consumed by the bacteria (Characklis and Marshall, 1990).

After 60 days of exposure of the metal samples in the industrial system, SEM analysis of the coupons with biofilms revealed the presence of varied bacterial cells, extracellular material, microbial filaments, and corrosion deposits (Figure 4).

SEM analysis of the coupons after the adhered biofilms were scraped off revealed signs of biocorrosion on the metal surface (Figure 5). This fact was to be expected due to the presence of aerobic and anaerobic microorganisms, including SRB, in the biofilm. According to Freedman (1993), microbiologically-influenced corrosion is not solely related to the presence of SRB and their metabolism; other forms of anaerobic, facultative, and aerobic bacteria can contribute to MIC on many different metals and in many different environments. The microorganisms can cause corrosion process by a lot of mechanisms, such as, cathodic depolarisation and excretion of acids metabolics.

Stereoscopic microscopic analysis of the samples exposed to sterile seawater under laboratory conditions during the same period of exposure to the industrial system did not detect any action of the salt water upon the metal surface.

The time of 60 days of exposure of the brass cupons in the industrial cooling water systems was enough to create a biofilm on the metal surface promoting the corrosion. This fact indicated that microbial activity in the corrosion process.

Figure 1: SEM microphotograph showing microfouling on the brass surface with colonization by different-shaped algae. Fifteen days of exposure. (Bar = 10 µm)

Figure 2: SEM microphotograph showing microfouling detail with colonization by algae and bacteria. Fifteen days of exposure. (Bar = 1 µm)

Figure 3: SEM microphotograph showing microfouling detail with colonization by algae serving as support for bacteria. Fifteen days of exposure. (Bar = 1µm)

Figure 4: SEM microphotograph showing microfouling on the brass surface with the presence of bacteria, filaments, EPS, and corrosion products. Sixty days of exposure. (Bar = 1µm)

Figure 5: SEM microphotograph of the brass coupon after the biofilm was scraped off the metal, revealing corrosion of the metal. Sixty days of exposure. (Bar = 10µm)

ACKNOWLEDGMENTS

The authors wish to thank the CNPq for financial support, the Duque de Caxias Refinery (REDUC) - PETROBRÁS for the use of their industrial system, and the CENPES - PETROBRÁS for the use of their SEM equipment.

REFERENCES

American Public Health Association (APHA), Tentative Methods of Analysis for Hydrogen Sulfide Content of the Atmosphere. In: Methods of Air Sampling and Analysis, pp. 426-432. Washington, D.C. (1972).

American Public Health Association (APHA), Standard Methods for Examination of Water and Waste Water, pp. 9.78-9.83. Washington, D.C. (1992).

Characklis, W.G. and Marshall, K.C., Biofilms (Characklis, W. G. and Marshall, K. C., ed.), John Wiley & Sons, New York, 796p. (1990).

Cook, P.E. and Gaylarde, C.C., Biofilms Formation in Aqueous Metal Working Fluids. Intern. Biodet. 24:265-270 (1988).

Coutinho, C.M.L.M.; Magalhães, F.C.M. and Jorge, T.C.A., Scanning Electron Microscope Study of Biofilm Formation at Different Flow Rates over Metal Surfaces Using Sulfate-Reducing Bacteria. Biofouling 7: 19-27 (1993).

França, F.P. de and Lutterbach, M.T.S., Variation in Sessile Microflora During Biofilm Formation on AISI-304 Stainless Steel Coupons. Journal of Industrial Microbiology 16: 1-5 (1996).

Elboujdaini, M. and Sastri, V.S., Field Studies of Microbiological Corrosion in Water Injection Plant. Corrosion, Paper nº213, Texas: NACE (1995).

Freedman, A.J., Microbiologically-Influenced Corrosion: A State-of-the-Art Technology. Corrosion, Paper nº635, Texas: NACE (1993).

Holt, J.G.; Krieg, N.R.; Sneath, P.H.A.; Stanley, J. T. and Williams, S.T. (eds.), Bergey’s Manual of Determinative Bacteriology, 9th ed. Williams & Wilkins Co., Baltimore, 787p. (1994).

Koch, A.L. Growth Measurement, In: Manual of Methods for General Bacteriology (Gerhardt, P., ed.), pp. 188-207. American Society for Microbiology, Washington, D.C. (1981).

Lee, W.; Lewandowski, A.; Nielsen, P. and Hamilton, A.W., Role of Sulfate-Reducing Bacteria in Corrosion of Mild Steel: A Review. Biofouling, 8: 165-194 (1995).

Lewandowski, Z.; Stoodley, P. and Roe, F., Internal Mass Transport in Heterogeneous Biofilms: Recent Advances. Corrosion, Paper nº222, Texas: NACE (1995).

Licina, G.J., Microbiologically-Influenced Corrosion in Nuclear Power Plants - A Review of Susceptibility and Mitigation Methods. Corrosion, Paper nº527, Texas: NACE (1990).

Lutterbach, M.T.S. and França, F.P. de, Biofilm Formation in Water Cooling Systems. World Journal of Microbiology & Biotechnology. 12:4, 391-394 (1996).

National Association of Corrosion Engineers (NACE), The Role of Bacteria in the Corrosion of Oil Field Equipment. In: Technical Practices Committee nº3. Houston, Texas (1976).

Page, S. and Gaylarde, C.C., Biocide Activity against Legionella & Pseudomonas. Intern. Biodet. 26: 139-148 (1990).

Postgate, J.R., The Sulfate-Reducing Bacteria. Cambridge University Press, Cambridge (1984).

Rosemberg, J.A., O potencial das algas. Possibilidades de Scenedesmus quadricauda isolada no Rio de Janeiro. Ph.D. Thesis, Universidade Federal do Rio de Janeiro, Brazil (1976).

Stoecker II, J.G., Microbiological Influence and Electrochemical Types of Corrosion: Back to Basics. Corrosion, Paper nº259, Texas: NACE (1994).

Soebbing, J.B. and Yolo, R.A, Microbiologically-Influenced Corrosion in Wastewater Treatment Plants. Corrosion, Paper nº214, Texas: NACE (1995).

Zisson, P.S.; Whitaker, J.M.; Neilson, H.L. and Mayne, L.L., Experiences with Monitoring and Control of Microbiological Growth in the Standby Service Water System of a BWR Nuclear Power Plant. Corrosion, Paper nº 264, Texas: NACE (1995).

Publication Dates

• Publication in this collection
  06 Oct 1998
• Date of issue
  Mar 1997

History

• Accepted
  30 Jan 1997
• Received
  06 Aug 1996